Treatment of myelodysplastic syndromes with 2-o and, or 3-o desulfated heparinoids

ABSTRACT

Methods are presented for treating cancers and hematopoietic stem cell disorders, comprising administering to a subject with a cancer or hematopoietic stem cell disorder who is receiving a treatment regimen, a heparin derivative capable of inhibiting, reducing, abrogating or otherwise interfering with the binding of CXCL12 to CXCR4, wherein the cancer or hematopoietic stem cell disorder is one in which interaction of CXCL12 with CXCR4 privileges the cancer or disordered HSCs against therapeutic intervention. In preferred embodiments, the heparin derivative is a substantially 2-O, 3-O-desulfated heparin derivative.

1. CROSS-REFERENCE TO RELATED APPLICATIONS

This is a continuation of U.S. patent application Ser. No. 15/044,740filed on Feb. 16, 2016, which claims priority to U.S. Provisional PatentApplication Nos. 62/277,360, filed Jan. 11, 2016; 62/181,513, filed Jun.18, 2015; and 62/117,409, filed Feb. 17, 2015, the disclosures of whichare incorporated herein by reference in their entireties for allpurposes.

2. BACKGROUND

Animal models have long predicted that heparin and various heparinderivatives would enhance the efficacy of chemotherapy in the treatmentof human cancers. Tani and colleagues, for example, reported thatheparin enhances potency of gemcitabine in a pancreatic cancer model(Tani et al., Abstract 4175, Cancer Res. 70(8) (Suppl. 1) (2010)). WO2012/106379 analogously reports that a substantially non-anticoagulating2-O, 3-O-desulfated heparin derivative, “ODSH”, improves the efficacy ofa chemotherapy regimen that includes gemcitabine in a standard tumorxenograft animal model of human pancreatic cancer.

However, the animal models have proven to be poor at predicting efficacyin human patients, and convincing evidence is sparse that adding heparinor various heparin derivatives enhances efficacy of standardantineoplastic treatment regimens.

For example, despite a wealth of data from both preclinical animalmodels and early human phase II trials that had suggested that lowmolecular weight heparin (“LMWH”) significantly prolongs survival in awide variety of cancers in patients without venous thromboembolism,Maraveyas and colleagues reported in 2012 that adding the LMWHdalteparin to gemcitabine provided no statistically significantimprovement in survival in advanced pancreatic cancer in a properlypowered trial (Maraveyas et al., Eur. J. Cancer 48:1283-1292 (2012)). Ina contemporaneous phase II randomized study, dalteparin could not beshown to improve outcome in ovarian cancer patients being treated with astandard chemotherapy regimen (Elit et al., Thromb Res. 130(6):894-900(2012)). A few months earlier, van Doormaal et al. had analogouslyreported that adding the LMWH nadroparin to existing standard of careprotocols in patients with advanced prostate, lung, or pancreatic cancerprovided no statistically significant survival benefit (van Doormaal etal., J. Clin. Oncol. 29:2071-2076 (2011)).

Similarly, despite the evidence from animal models that ODSH couldenhance the efficacy of chemotherapeutic regimens in treatment ofpancreatic cancer, no statistically significant benefit inprogression-free survival or overall survival was observed in a laterhuman clinical trial testing addition of ODSH to the standard-of-carechemotherapy regimen of gemcitabine plus nab-paclitaxel in patients withmetastatic adenocarcinoma of the pancreas (Clinical Trial.govNCT01461915).

Despite continuing advances in treating cancer, there is still a need inthe art for more effective treatments, and for treatments that havefewer side effects.

Myelodysplastic syndromes (“MDS”) represent a spectrum of clonalhematopoietic stem cell disorders characterized by progressive bonemarrow failure and increased risk of progression to acute myeloidleukemia (“AML”, also known as “acute myelogenous leukemia”). TheInternational Prognostic Scoring System (“IPSS”) is widely used toidentify patients with high risk features based on the severity of theircytopenias, bone marrow myeloblast percentage, and cytogeneticabnormalities. For patients with MDS, allogeneic hematopoietic stem celltransplantation remains the only curative treatment option. However, MDSis a disease of older individuals, with fewer than 5 percent of casesoccurring in patients younger than 50 years and the majority beingdiagnosed at an age over 70 years. Because of age, comorbidities, andother factors , less than 10 percent of all MDS patients are able toproceed to potentially curative allogeneic hematopoietic stem celltransplantation.

Hypomethylating agents are considered standard first line therapy forpatients with higher risk disease. Unfortunately, these agents are notcurative and only achieve remission in approximately 20-30 percent ofpatients, with a median duration of response of 8-10 months. Outcomesafter hypomethylating agents are poor. There remains an unmet need forbetter treatment of myelodysplasias.

3. SUMMARY

It has now been discovered that heparin derivatives (collectively,“heparinoids”) that are capable of inhibiting, reducing, abrogating orotherwise interfering with the binding of CXCL12 to CXCR4(“CXCL12-interacting heparinoids”) can increase the efficacy ofantineoplastic regimens against a selected subset of cancers, those inwhich interaction of CXCL12 with CXCR4 privileges the cancer againsttherapeutic intervention. In certain embodiments, the cancers are thosein which neoplastic cells, including but not limited to cancer stemcells, migrate to and/or reside in one or more anatomic sites, such asthe bone marrow, that provide protection from the antineoplasticregimen. In certain embodiments, the cancers are those in which stromalcell expression of CXCL12 protein exerts a prosurvival influence ontumor cells.

Moreover, because the bone marrow niche upregulates production of CXCR4and CXCR12 in disorders of hematopoietic stem cells (“HSC”s), and thisupregulation is believed to promote survival of the disordered HSCs,heparinoids that are capable of inhibiting, reducing, abrogating orotherwise interfering with the binding of CXCL12 to CXCR4 can increasethe efficacy of agents, such as hypomethylating agents, such asazacitidine, that are used to treat such HSC disorders, such as MDS.

Thus, in a first aspect, methods of treating cancer are provided.

The methods comprise administering to a subject receiving anantineoplastic treatment regimen a heparin derivative capable ofinhibiting, reducing, abrogating or otherwise interfering with thebinding of CXCL12 to CXCR4, wherein the cancer is one in whichinteraction of CXCL12 with CXCR4 privileges the cancer againsttherapeutic intervention. The heparin derivative is administered in anamount and at a time effective to enhance effectiveness of theantineoplastic treatment regimen.

In certain embodiments, the cancer is one in which neoplastic cells,such as cancer stem cells, migrate to and/or reside in anatomic sitesthat are capable of protecting the neoplastic cells from theantineoplastic treatment regimen. The heparin derivative is administeredin an amount and at a time effective to enhance effectiveness of theantineoplastic treatment regimen. In typical embodiments, the heparinderivative is administered in an amount effective to mobilize neoplasticcells from the anatomic site that is capable of protecting theneoplastic cells from the antineoplastic treatment regimen. Typically,the amount is effective to mobilize neoplastic cells from the bonemarrow.

In certain embodiments, the cancer is one in which stromal cellexpression of CXCL12 protein exerts a prosurvival influence on tumorcells. The heparin derivative is administered in an amount and at a timeeffective to enhance effectiveness of the antineoplastic treatmentregimen. In typical embodiments, the heparin derivative is administeredin an amount effective to reduce CXCL12-CXCR4 interaction. In certainembodiments, the heparin derivative is administered in an amounteffective to reduce tumor-specific immunosuppression.

In a related aspect, improved methods are provided for treating cancerswith an antineoplastic treatment regimen, wherein the cancer is one inwhich interaction of CXCL12 with CXCR4 privileges the cancer againsttherapeutic intervention, the improvement comprising furtheradministering a heparin derivative that is capable of inhibiting,reducing, abrogating, or otherwise interfering with the binding ofCXCL12 to CXCR4, in an amount and at a time effective to enhanceeffectiveness of the antineoplastic treatment regimen.

In certain embodiments, the cancer is one in which neoplastic cellsmigrate to and/or reside in anatomic sites capable of protecting theneoplastic cells from an antineoplastic treatment regimen, theimprovement comprising further administering a heparin derivative thatis capable of inhibiting, reducing, abrogating, or otherwise interferingwith the binding of CXCL12 to CXCR4, in an amount and at a timeeffective to enhance effectiveness of the antineoplastic treatmentregimen. In typical embodiments, the heparin derivative is administeredin an amount effective to mobilize neoplastic cells from the anatomicsite that is capable of protecting the neoplastic cells from theantineoplastic treatment regimen. Typically, the amount is effective tomobilize neoplastic cells from the bone marrow.

In certain embodiments, the cancer is one in which stromal cellexpression of CXCL12 protein exerts a prosurvival influence on tumorcells, the improvement comprising further administering a heparinderivative that is capable of inhibiting, reducing, abrogating orotherwise interfering with the binding of CXCL12 to CXCR4, in an amountand at a time effective to enhance effectiveness of the antineoplastictreatment regimen. In typical embodiments, the heparin derivative isadministered in an amount effective to reduce CXCL12-CXCR4 interaction.In certain embodiments, the heparin derivative is administered in anamount effective to reduce tumor-specific immunosuppression.

In certain embodiments, the cancer is a carcinoma. In certainembodiments, the cancer is lung cancer. In certain other embodiments,the lung cancer is non-small-cell lung cancer. In certain otherembodiments, the cancer is a hematologic cancer. In certain embodiments,the hematologic cancer is leukemia. In certain other embodiments,leukemia is acute myeloid leukemia (AML). In certain embodiments the AMLis primary AML. In certain other embodiments, the AML is secondary AML.

In another aspect, methods of treating disorders of hematopoietic stemcells are provided. The methods comprise administering to a subjectreceiving a treatment regimen for a hematopoietic stem cell disorder aheparin derivative capable of inhibiting, reducing, abrogating orotherwise interfering with the binding of CXCL12 to CXCR4. The heparinderivative is administered in an amount and at a time effective toenhance effectiveness of the treatment regimen.

In certain embodiments, the hematopoietic stem cell disorder is one inwhich the disordered HSC cells migrate to and/or reside in one or moreanatomic sites that provide protection from the treatment regimen, suchas the bone marrow. In certain embodiments, the hematopoietic stem celldisorder is one in which stromal cell expression of CXCL12 proteinexerts a prosurvival influence on the disordered HSCs.

In some embodiments, the disordered HSC cells have abnormal karyotype.In some of these embodiments, the disordered HSC cells are pre-cancerousstem cells.

In certain embodiments, the hematopoietic stem cell disorder is MDS. Incertain embodiments, the disorder is newly diagnosed MDS. In certainembodiments, the disorder is recurrent or refractory MDS.

In certain embodiments, the subject has been diagnosed with MDS andsymptomatic anemia. In some of these embodiments, the subject hashemoglobin levels less than 10.0 g/dL or requires red blood celltransfusion. In certain embodiments, the subject has been diagnosed withMDS and thrombocytopenia. In some of these embodiments, the subject hasa history of two or more platelet counts less than 50,000/μL or asignificant hemorrhage requiring platelet transfusions. In certainembodiments, the subject has been diagnosed with MDS and neutropenia. Insome of these embodiments, the subject has two or more absoluteneutrophil counts less than 1,000/μL. In certain embodiments, thesubject has been diagnosed with MDS and has an IPSS score of INT-1 orhigher prior to treatment.

In certain embodiments, the treatment regimen is hypomethylationtherapy.

In certain embodiments, the subject has undergone greater than or equalto 4 cycles of treatment of a hypomethylating agent without response, orhave documented disease progression after prior response to ahypomethylating therapy.

In certain embodiments, the hypomethylation agent is decitabine.

In certain embodiments, the hypomethylation agent is azacitidine.

In certain embodiments, the azacitidine is administered to the subjectintravenously.

In certain embodiments, the azacitidine is administered at a dosagerange of 5-500 mg/m².

In certain embodiments, the azacitidine is administered at 75 mg/m² as a15 minute intravenous infusion daily on days 1 through 5 of each 28-daycycle.

In certain embodiments, the azacitidine is administered for up to 6cycles.

In certain embodiments, the heparin derivative is administered to thesubject intravenously.

In certain embodiments, the heparin derivative is administeredcontinuously.

In certain embodiments, the heparin derivative is administered as abolus injection.

In certain embodiments, the heparin derivative is administered as abolus injection followed by continuous administration.

In certain embodiments, the heparin derivative is administeredsubcutaneously.

In certain embodiments, the heparin derivative is administered at adosage range of 0.01 mg/kg to 100 mg/kg.

In certain embodiments, the heparin derivative is administered as a 4mg/kg bolus on Day 1 followed by a continuous intravenous infusion of0.25 mg/kg/hr for days 1 through 5 of each 28-day cycle.

In certain embodiments, the heparin derivative is administered for up to6 cycles.

In certain embodiments, the heparin derivative is administered prior tothe antineoplastic treatment.

In certain embodiments, the heparin derivative is administeredconcurrently with the antineoplastic treatment.

In certain embodiments, the heparin is administered prior to andconcurrently with the antineoplastic treatment.

In certain embodiments, the disorder is one in which neoplastic cells orpre-neoplastic cells, such as cancer stem cells, migrate to and/orreside in anatomic sites that are capable of protecting the neoplasticor pre-neoplastic cells from the antineoplastic treatment regimen. Theheparin derivative is administered in an amount and at a time effectiveto enhance effectiveness of the antineoplastic treatment regimen. Intypical embodiments, the heparin derivative is administered in an amounteffective to mobilize neoplastic cells from the anatomic site that iscapable of protecting the neoplastic cells or pre-neoplastic cells fromthe antineoplastic treatment regimen. Typically, the amount is effectiveto mobilize neoplastic cells from the bone marrow.

In certain embodiments, the amount of the heparin derivative iseffective to cause a complete response or a near complete response ratein the subject.

In certain embodiments, the amount is effective to cause a partialresponse rate in the subject.

In certain embodiments, performing the methods will determine thetolerability and toxicities of combination treatment of azacitidine andazacitidine and heparin derivatives.

In certain embodiments, performing the methods will determine the eventfree, progression free, disease free, 10 year survival and overallsurvival of subjects treated with azacitine and azacitidine and heparinderivatives.

In certain embodiments, performing the methods will determinehematologic improvement evaluated by absolute neutrophil count, plateletand red blood cell response.

In certain embodiments, performing the methods will determinecytogenetic response as evaluated by reversion to normal karyotype.

In certain embodiments, the disorders are hematologic disorders.

In certain embodiments, the disorders are those in which cells arepre-neoplastic cells.

In certain embodiments, the disorder is myelodysplastic syndrome (MDS).

In certain embodiments, the disorder is newly diagnosed MDS.

In certain embodiments, the disorder is recurrent or refractory MDS.

In certain embodiments, the disorders are those in which stromal cellexpression of CXCL12 protein exerts a prosurvival influence on thecells.

In certain embodiments, the anti-neoplastic regiment is treatment with ahypomethylating agent.

In certain embodiments, the hypomethylating agent is azacitidine.

4. BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the chemical formula of the ATIII-binding pentasaccharidesequence of USP heparin (also known as “unfractionated heparin”, or“UFH”) and the comparable sequence of a 2-O, 3-O-desulfated heparinderivative prepared by cold alkaline hydrolysis of UFH.

FIGS. 2A-2C are photomicrographs of serial bone marrow biopsies of apatient with acute myeloid leukemia (“AML”, also known as acutemyelogenous leukemia) treated with an ODSH pharmaceutical composition(“CX-01”) in combination with cytarabine and idarubicin, as described inExample 1. FIG. 2A shows bone marrow prior to treatment. FIG. 2B showsmarrow at day 14 of treatment. FIG. 2C shows day 28 marrow.

FIG. 3 compares the complete response rate (CR) observed in the clinicaltrial described in Example 1 to historical control (indicated byasterisk).

5. DETAILED DESCRIPTION 5.1. Overview of Experimental Observations

Data from animal models have long suggested that heparin and certainheparin derivatives can enhance the efficacy of antineoplastic treatmentregimens, such as chemotherapy. Despite sporadic anecdotal reports inhuman patients, however, there is sparse evidence of statisticallysignificant clinical enhancement in human cancer patients.

For example, the substantially non-anticoagulant 2-O, 3-O desulfatedheparin derivative, ODSH (see FIG. 1), was shown to sensitize cancercells to chemotherapy in animal models of human pancreaticadenocarcinoma (WO 2012/106379). When later tested in a human clinicaltrial (ClinicalTrials.gov identifier NCT01461915), however, addition ofODSH to the chemotherapy regimen did not provide a statisticallysignificant increase in progression-free survival or overall survival inpatients with metastatic adenocarcinoma of the pancreas.

Although unable to prolong progression-free survival or overall survivalin patients with metastatic pancreatic cancer, ODSH was found,unexpectedly, to attenuate the myelosuppressive side effects of thegemcitabine plus nab-paclitaxel chemotherapy regimen (U.S. Pat. No.8,734,804, incorporated herein by reference in its entirety).

To confirm the myeloprotective effects of ODSH, a second clinical trialwas initiated in a different cancer, acute myeloid leukemia (“AML”, alsoknown as acute myelogenous leukemia), treated with a differentmyelosuppressive chemotherapeutic regimen, idarubicin plus cytarabine(ClinicalTrials.gov identifier: NCT02056782).

As described in detail in Example 1, below, ODSH significantlyattenuated the myelosuppressive side effects of theidarubicin+cytarabine anti-AML chemotherapy regimen, as expected. Inaddition, however, and unexpectedly given prior failure of ODSH toimprove response to chemotherapy in the pancreatic cancer trial, ODSHalso improved the efficacy of the chemotherapy treatment: 11 out of 12patients (92%) treated with both ODSH and idarubicin plus cytarabine,including two patients who received an incomplete course of chemotherapy(3 and 5 days respectively), had a morphologic complete remission at theend of a single induction cycle, higher than would otherwise have beenexpected. All 11 patients with primary AML achieved a complete remissionat the end of a single induction cycle. Furthermore, 10 of the 12patients remain in complete remission 5-13 months after having beenenrolled in the study.

Thus, it has now been discovered that ODSH can increase the efficacy ofantineoplastic regimens against a selected subset of cancers,notwithstanding the fact that ODSH cannot increase the efficacy ofantineoplastic regimens against various other cancers.

As described in Example 2, serial bone marrow biopsies drawn from one ofthe patients treated with ODSH, cytarabine, and idarubicin in the AMLclinical trial unexpectedly showed significant depletion of cellularelements in addition to the expected depletion of leukemic cells. FIG.2A shows bone marrow prior to treatment, demonstrating that the marrowis packed with leukemia cells. FIG. 2B shows marrow at Day 14 of theinduction cycle, demonstrating elimination of most normal bone marrowcells as well as leukemia cells. FIG. 2C shows day 28 marrow, with noevidence of leukemic cells and restoration of normal bone marrowappearance and function.

Without intending to be bound by theory, the unexpected clearance ofcells from the marrow seen in the Day 14 biopsy suggests that theincreased remission rate observed in the AML clinical trial can beattributed to ODSH-mediated mobilization of leukemic cells from themarrow into the peripheral circulation, where they became vulnerable tothe infusions of cytarabine and idarubicin. Retention of leukemic cellsin the bone marrow is known to make them more resistant to chemotherapy(Hope et al., Nat. Immunol. 5:738-742 (2004)). The recovery of themarrow by Day 28 demonstrates further that the ODSH-mediated flushing ofcells from the marrow does not adversely affect the ability of themarrow to repopulate and support multi-lineage hematopoiesis. Indeed,the accelerated recovery of platelet and white cell count, consistentwith observations from the previous trial in pancreatic cancer,demonstrates that the marrow microenvironments required forthrombopoiesis, erythropoiesis, and granulopoiesis remain healthy.

CXCL12, also known as Stromal Cell Derived Factor-1 or SDF-1, wasoriginally described as a CXC chemokine produced locally within the bonemarrow compartment to provide a homing signal for hematopoietic stemcells (“HSC”s). CXCL12 is the ligand for the CXCR4 receptor on thesurface of HSCs; ligation of CXCR4 by CXCL12 is known to promote stemcell survival, proliferation, migration, and chemotaxis (see, e.g.,Lapidot et al., Leukemia 16(10):1992-2003 (2002)). It has also beenreported that the CXCR4 receptor is prominently expressed on the cellmembrane of many cancer cells, particularly cancer stem cells (Yu etal., Gene 374:174-9 (2006); Cojoc et al., Oncotargets & Therapy6:1347-1361(2013)), and that the CXCL12/CXCR4 interaction may mediatemigration of cancer cells to anatomic sites that produce CXCL12 (Wald etal., Theranostics 3:26-33 (2013); Cojoc et al., supra).

5.2. Methods of Treatment 5.2.1. Methods of Treating Cancer

Accordingly, in a first aspect, methods of treating cancer are provided.

The methods comprise administering to a subject receiving anantineoplastic treatment regimen a heparin derivative capable ofinhibiting, reducing, abrogating or otherwise interfering with thebinding of CXCL12 to CXCR4, wherein the cancer is one in whichinteraction of CXCL12 with CXCR4 privileges the cancer againsttherapeutic intervention. The heparin derivative is administered in anamount and at a time effective to enhance effectiveness of theantineoplastic treatment regimen.

In certain embodiments, the cancer is one in which neoplastic cells,such as cancer stem cells, migrate to and/or reside in anatomic sitesthat are capable of protecting the neoplastic cells from theantineoplastic treatment regimen. The heparin derivative is administeredin an amount and at a time effective to enhance effectiveness of theantineoplastic treatment regimen. In typical embodiments, the heparinderivative is administered in an amount effective to mobilize neoplasticcells from the anatomic site that is capable of protecting theneoplastic cells from the antineoplastic treatment regimen. Typically,the amount is effective to mobilize neoplastic cells from the bonemarrow.

In certain embodiments, the cancer is one in which stromal cellexpression of CXCL12 protein exerts a prosurvival influence on tumorcells. The heparin derivative is administered in an amount and at a timeeffective to enhance effectiveness of the antineoplastic treatmentregimen. In typical embodiments, the heparin derivative is administeredin an amount effective to reduce CXCL12-CXCR4 interaction. In certainembodiments, the heparin derivative is administered in an amounteffective to reduce tumor-specific immunosuppression.

In a related aspect, improved methods are provided for treating cancerswith an antineoplastic treatment regimen, wherein the cancer is one inwhich interaction of CXCL12 with CXCR4 privileges the cancer againsttherapeutic intervention, the improvement comprising furtheradministering a heparin derivative that is capable of inhibiting,reducing, abrogating or otherwise interfering with the binding of CXCL12to CXCR4, in an amount and at a time effective to enhance effectivenessof the antineoplastic treatment regimen.

In certain embodiments, the cancer is one in which neoplastic cellsmigrate to and/or reside in anatomic sites capable of protecting theneoplastic cells from an antineoplastic treatment regimen, theimprovement comprising further administering a heparin derivative thatis capable of inhibiting, reducing, abrogating or otherwise interferingwith the binding of CXCL12 to CXCR4, in an amount and at a timeeffective to enhance effectiveness of the antineoplastic treatmentregimen. In typical embodiments, the heparin derivative is administeredin an amount effective to mobilize neoplastic cells from the anatomicsite that is capable of protecting the neoplastic cells from theantineoplastic treatment regimen. Typically, the amount is effective tomobilize neoplastic cells from the bone marrow.

In certain embodiments, the cancer is one in which stromal cellexpression of CXCL12 protein exerts a prosurvival influence on tumorcells, the improvement comprising further administering a heparinderivative that is capable of inhibiting, reducing, abrogating orotherwise interfering with the binding of CXCL12 to CXCR4, in an amountand at a time effective to enhance effectiveness of the antineoplastictreatment regimen. In typical embodiments, the heparin derivative isadministered in an amount effective to reduce CXCL12-CXCR4 interaction.In certain embodiments, the heparin derivative is administered in anamount effective to reduce tumor-specific immunosuppression.

5.2.1.1. Selected Cancers 5.2.1.1.1. Cancers Characterized by Migrationto Privileged Anatomic Sites

In certain embodiments of the methods described herein, the cancer isselected from those in which neoplastic cells migrate to and/or residein anatomic sites that are capable of protecting the neoplastic cellsfrom an antineoplastic treatment regimen (hereinafter, “privilegedanatomic sites”). In various embodiments, the privileged anatomic siteis selected from the group consisting of bone marrow, liver, and brain.In typical embodiments, the privileged anatomic site is the bone marrow.

In some embodiments, the cancer is a hematologic cancer. In various suchembodiments, the cancer is selected from the group consisting of acutemyeloid leukemia (“AML”, also known as acute myelogenous leukemia, or“AML”), acute lymphoblastic leukemia, chronic lymphocytic leukemia,chronic myelogenous leukemia, and acute monocytic leukemia.

In some embodiments, the cancer to be treated is a cancer havingsubstantial potential to metastasize to the bone marrow. In various ofthese embodiments, the cancer is selected from the group consisting ofprostate cancer, breast cancer, lung cancer and melanoma, all of whichshow high rates of metastasis to the bone and can home into the nicheoccupied by HSCs (see, e.g., Kaplan et al., Cancer Metastasis Rev.25(4):521-9 (2006), incorporated herein by reference in its entirety).In certain embodiments, the cancer is kidney cancer, thyroid cancer, orneuroblastoma. In certain embodiments, the cancer to be treated is headand neck cancer, esophagus cancer, stomach cancer, colorectal cancer, orsarcoma.

In some embodiments, the cancer to be treated is selected from the groupconsisting of metastatic prostate cancer, metastatic lung cancer,including metastatic non-small cell lung cancer, metastatic breastcancer, and metastatic neuroblastoma.

In some embodiments, the cancer to be treated is lung cancer.

In certain lung cancer embodiments, the cancer is small cell lungcancer. In other embodiments, the lung cancer is non-small cell lungcancer (“NSCLC”).

In certain NSCLC embodiments, the NSCLC is locally advanced. In certainembodiments, the NSCLC is inoperable. In certain embodiments, the NSCLCis locally advanced and inoperable. In certain embodiments, thenon-small cell lung cancer is Stage IIIB NSCLC. In some embodiments, theNSCLC is oligometastatic stage IV non-small cell lung cancer. In someembodiments, the NSCLC is being treated with radiation therapy andchemotherapy.

In some embodiments, the cancer to be treated is characterized by thepresence of post-treatment minimal residual disease. “Minimal residualdisease” generally refers to cancer cells that persist afterantineoplastic therapy, and whose presence is correlated with relapse ofthe disease. Without intending to be bound by theory, the minimalresidual disease state is attributed to persistence of cancer stemcells, often resident in privileged anatomic sites, and which are moreresistant to therapeutic treatments and have the capacity to give riseto variant cancer cell types found in the particular cancer. By escapingeffect of cancer treatments, the cancer stem cells can cause relapse andmetastasis by producing new cancer cell types. In particular, cancerswith minimal residual disease in the bone marrow, liver, brain, or othersimilar tissues are appropriate cancers to be treated according to themethods described herein.

In certain embodiments, cancers with minimal residual disease states areselected from the group consisting of breast cancer, glioblastoma, smallcell lung cancer, non-small cell lung cancer, prostate cancer, primaryacute myeloid leukemia, secondary acute myeloid leukemia, refractoryacute myeloid leukemia, and chronic myelogenous leukemia. In someembodiments, any cancer with minimal residual disease in the bone marrowor like tissues that protect neoplastic cells from antineoplastictreatment following initial antineoplastic treatment is an appropriatecancer to be treated according to the methods described herein.

5.2.1.1.2. Cancers Characterized by Stromal Expression of CXCL12

In certain embodiments, the cancer is selected from those in whichstromal expression of CXCL12 protein exerts a prosurvival influence ontumor cells.

In certain embodiments, the cancer is selected from adenocarcinomas. Incertain embodiments, the cancer is selected from primary and metastaticcarcinomas. In various embodiments, the cancer is selected fromprostate, colorectal, breast, ovarian, bladder, lung (including smallcell lung cancer and non-small cell lung cancer), and hepatocellularcarcinoma.

5.2.1.2. Antineoplastic Treatment Regimens

In the methods described herein, the antineoplastic treatment regimen isany antineoplastic treatment regimen appropriate for the cancer beingtreated.

In some embodiments, the treatment regimen includes chemotherapy. Insome embodiments, the treatment regimen includes radiation therapy. Insome embodiments, the treatment regimen includes antibody therapy. Insome embodiments, the treatment regimen includes therapy targeted tomutant enzymes, such as mutated kinases. In some embodiments, thetreatment regimen includes immunotherapy, such as immunotherapy with acheckpoint inhibitor.

In the methods provided herein, the antineoplastic treatment regimen canbe myelosuppressive or non-myelosuppressive.

Myelosuppressive antineoplastic treatment regimens include those thatreduce one or more of platelet count, red blood cell count, white bloodcell count, and particularly, neutrophil count. In certain embodiments,the myelosuppressive antineoplastic treatment regimen is capable ofcausing a grade 1, grade 2, grade 3, or grade 4 thrombocytopenia whenadministered without adjunct administration of a CXCL12-interactingheparinoid. In some embodiments, the myelosuppressive antineoplastictreatment regimen is capable of causing a grade 1, grade 2, grade 3, orgrade 4 neutropenia when administered without adjunct administration ofa CXCL12-interacting heparinoid.

In some embodiments, the myelosuppressive antineoplastic treatmentregimen includes administration of one or more of an alkylating agent,antimetabolite, anthracyclines, topoisomerase inhibitors or mitoticinhibitors.

In some embodiments, the myelosuppressive antineoplastic treatmentregimen includes administration of one or more of venetoclax,decitabine, LY573636, aldesleukin, bortezomib, ixazomib, tipifarnib,panobinostat, pracinostat, clorfarabine, alvocidib, lenolidamide,dasatinib, volasertib, sorafenib, CP-351, vosaroxin, etoposide,mitoxantrone, guadecitabine, gemtuzumab ozogamicin, SGN-CD33A, BI836858, AGS67E, arsenic trioxide, vorinostat, binimetinib, trametinib,BVD-523, E6201, vyxeos, AZD1775, 8-chloro-adenosine, cladribine,flutarabine, capecitidine, pomalidomide, erwinaze, treosulfan,alisertib, gedatolisib, ruxolitinib, LY2606368, OXi4503, gliteritinib,sunitinib, lestaurtinib, midostaurin, quizartinib, crenolanib,pacritinib, AKN-028, FLX925 or E6201.

In some embodiments, the myelosuppressive antineoplastic treatmentregimen includes administration of one or more of a FMS-related tyrosinekinase-3 inhibitor, a tyrosine kinase inhibitor, a proteasome inhibitor,a histone deacetylase inhibitor, a CD-33 inhibitor, a MEK inhibitor, apurine analog, an asparaginase, an mTOR inhibitor or an Aurora Kinaseinhibitor.

In particular embodiments, the antineoplastic treatment regimen is anon-myelosuppressive treatment regimen. As used herein,“non-myelosuppressive” treatment regimen refers to a treatment regimenthat does not substantially reduce one or more of platelet count, redblood cell count, white blood cell count, and neutrophil count whenadministered without adjunct administration of a CXCL12-interactingheparinoid. In preferred embodiments, the non-myelosuppressive treatmentregimen does not cause a grade 1, grade 2, grade 3, or grade 4thrombocytopenia when administered without adjunct administration of aCXCL12-interacting heparinoid. In certain embodiments, thenon-myelosuppressive treatment regimen does not cause a grade 1, grade2, grade 3, or grade 4 neutropenia when administered without adjunctadministration of a CXCL12-interacting heparinoid.

In some embodiments, the non-myelosuppressive antineoplastic treatmentregimen includes administration of one or more of a kinase inhibitor, aVEGF inhibitor, a VEGFR inhibitor, a VEGFR2 inhibitor, a PDGFRinhibitor, a Src family kinase inhibitor, a hedgehog inhibitor, aretinoid X receptor activator, a histone methyltransferase inhibitor, aBCL2 inhibitor, an AKT inhibitor, a CXCR4 inhibitor, an mTOR inhibitor,an Mdm2 antagonist, an Mdm2 inhibitor, a CD25 inhibitor, a CD47inhibitor, an IL-3R inhibitor, a BCR-Abl inhibitor, a HSP90 inhibitor,an HGF inhibitor, a MET inhibitor and a bromodomain and extra-terminaldomain (BET) inhibitor and a BRD4 inhibitor.

In some embodiments, the non-myelosuppressive treatment regimen includesadministration of one or more of crizotinib, seliciclib, afatinib,aldesleukin, alemtuzumab; axitinib, belinostat, bosutinib, brentuximabvedotin, carfilzomib, ceritinib, dabrafenib, dasatinib, everolimus,ibritumomab tiuxetan, ibrutinib, sorafenib, idelalisib, ipilimumab,nilotinib, obinutuzumab, ofatumumab, panitumumab, pembrolizumab,pertuzumab, ponatinib, ramucirumab, regorafenib, romidepsin, sipuleucel,temsirolimus, tositumomab, trametinib, vandetanib, vemurafenib,vismodegib, vorinostat, ziv-aflibercept, cabozantinib, selinexnor,PF-4449913, erismodegib, GO-203-2C, thioridazine, nivolumab. bexarotene,EPZ-5676, ABT-199, GSK2141795, entospletinib, TAK-659, CPI-613, B1-8040,LY2510924, plerixafor, mozobil, OCV-501, pacritinib, eltrombopag,promacta, revolade, nintedanib, vargatef, rapamycin, MEN1112,ipilimumab, idasanutlin, R06839921, AMG-232, ADCT-301, KHK2823,CWP232291, SL-401, CC-90002, GSK2879552, lirilumab, BGB324, OTX-015,TEN-010, I-BET 762, CPI-203, CPI-0610, AG-120, AG-221 or IDH305.

In some embodiments, the non-myelosuppressive treatment regimen includesadministration of one or more of bleomycin, vincristine, prednisolone,and gallium nitrate.

In some embodiments, the non-myelosuppressive antineoplastic treatmentregimen comprises administration of a non-myelosuppressive targetedtherapeutic agent or an immunostimulatory or immunomodulatorytherapeutic agent. As used herein, a “non-myelosuppressive targetedtherapeutic” refers to a therapeutic agent that targets the cancer cellwith sufficient specificity to not have myelosuppressive effects.

A “non-myelosuppressive immunostimulatory therapeutic” refers to atherapeutic agent that stimulates immune activity against the cancercells, such as by reducing suppression of effector immune cells oractivating immune cells that result in a therapeutic response againstthe cancer cells.

In some embodiments, the immunostimulatory therapeutic agent comprisesone or more checkpoint inhibitors. In certain embodiments, thecheckpoint inhibitor is a monoclonal antibody. In certain embodiments,the monoclonal antibody is selected from an anti-CTLA-4 monoclonalantibody, an anti-PD1 monoclonal antibody, an anti-PDL1 monoclonalantibody, and combinations thereof.

5.2.2. Methods of Treating Hematopoietic Stem Cell Disorders

In another aspect, methods of treating hematopoietic stem cell disordersare provided.

The methods comprise administering to a subject receiving a treatmentregimen for a hematopoietic stem cell disorder a heparin derivativecapable of inhibiting binding of CXCL12 to CXCR4. The heparin derivativeis administered in an amount and at a time effective to enhanceeffectiveness of the treatment regimen.

In certain embodiments, the hematopoietic stem cell disorder is one inwhich the disordered HSC cells migrate to and/or reside in one or moreanatomic sites that provide protection from the treatment regimen, suchas the bone marrow. In certain embodiments, the hematopoietic stem celldisorder is one in which stromal cell expression of CXCL12 proteinexerts a prosurvival influence on the disordered HSCs. In certainembodiments, the hematopoietic stem cell disorder is one in whichstromal cell expression of CXCL12 protein inhibits apoptosis of thedisordered HSCs.

In some embodiments, the disordered HSC cells have abnormal karyotype.In some of these embodiments, the disordered HSC cells are pre-cancerousstem cells.

In certain embodiments, the hematopoietic stem cell disorder ismyelodysplastic syndrome (“MDS”, also known as “myelodysplasia”). Incertain embodiments, the disorder is newly diagnosed MDS. In certainembodiments, the disorder is recurrent or refractory MDS.

In certain embodiments, the subject has been diagnosed with MDS andsymptomatic anemia. In some of these embodiments, the subject hashemoglobin levels less than 10.0 g/dL or requires red blood celltransfusion. In certain embodiments, the subject has been diagnosed withMDS and thrombocytopenia. In some of these embodiments, the subject hasa history of two or more platelet counts less than 50,000/μL or asignificant hemorrhage requiring platelet transfusions. In certainembodiments, the subject has been diagnosed with MDS and neutropenia. Insome of these embodiments, the subject has two or more absoluteneutrophil counts less than 1,000/μL. In certain embodiments, thesubject has been diagnosed with MDS and has an IPSS score of INT-1 orhigher prior to treatment.

In typical embodiments, the treatment regimen for myelodysplasia ishypomethylation therapy. In certain embodiments, the subject has notundergone prior treatment with hypomethylation therapy. In otherembodiments, the subject has undergone prior treatment with ahypomethylating agent. In various of these embodiments, the subject hasundergone 1 prior treatment, 2 prior treatments, 3 prior treatments, oreven 4 prior treatments with a hypomethylating agent without completeremission.

In certain embodiments, the hypomethylation agent is decitabine.

In certain embodiments, the hypomethylation agent is azacitidine. Incertain azacitidine embodiments, the azacitidine is administered to thesubject intravenously. In certain embodiments, the azacitidine isadministered at a dosage range of 5-500 mg/m². In certain embodiments,the azacitidine is administered at 75 mg/m² as a 15 minute intravenousinfusion daily on days 1 through 5 of each 28-day cycle. In certainembodiments, the azacitidine is administered for up to 6 cycles.

In certain embodiments, the heparin derivative is administered to thesubject intravenously. In certain intravenous embodiments, the heparinderivative is administered as a bolus injection. In certain intravenousembodiments, the heparin derivative is administered continuously. Incertain intravenous embodiments, the heparin derivative is administeredas a bolus injection followed by continuous administration.

In certain embodiments, the heparin derivative is administered as a 4mg/kg bolus on Day 1 followed by a continuous intravenous infusion of0.25 mg/kg/hr for days 1 through 5 of each 28-day cycle. In certainembodiments, the heparin derivative is administered for up to 6 cycles.In certain embodiments, the heparin derivative is administered prior tothe myelodysplasia treatment regimen. In certain embodiments, theheparin derivative is administered concurrently with the myelodysplasiatreatment regimen. In certain embodiments, the heparin is administeredprior to and concurrently with the myelodysplasia treatment regimen. Intypical embodiments, the myelodysplasia treatment regimen isadministration of a hypomethylation agent.

In certain embodiments, the heparin derivative is administeredsubcutaneously. In certain subcutaneous administration embodiments, theheparin derivative is administered at a dosage range of 0.01 mg/kg to100 mg/kg.

In certain embodiments, the hematopoietic stem cell disorder is one inwhich the disordered HSC cells migrate to and/or reside in anatomicsites that are capable of protecting the cells from the treatmentregimen. The heparin derivative is administered in an amount and at atime effective to enhance effectiveness of the treatment regimen. Intypical embodiments, the heparin derivative is administered in an amounteffective to mobilize HSC cells from the anatomic site that is capableof protecting the disordered HSC cells from the antineoplastic treatmentregimen. Typically, the amount is effective to mobilize HSC cells fromthe bone marrow.

In certain embodiments, the amount of the heparin derivative iseffective to cause a complete response or a near complete response ratein the subject. In certain embodiments, the amount is effective to causea partial response rate in the subject.

In certain embodiments, the methods of treatment are effective to resultin improved event free survival as compared to treatment withoutadministration of the heparin derivative. In some embodiments, themethods of treatment are effective to result in improved progressionfree survival as compared to treatment without administration of theheparin derivative. In some embodiments, the methods of treatment areeffective to result in improved disease free survival, 10 year survival,and/or overall survival as compared to treatment without administrationof the heparin derivative. In certain embodiments, the methods oftreatment result in reversion of the disordered HSC cells to normalkaryotype.

5.2.3. Effective Heparin Derivatives

In the methods described herein, the heparin derivative is one capableof inhibiting, reducing, abrogating, or otherwise interfering with thebinding of CXCL12 to CXCR4. For convenience, such heparin derivativesare collectively referred to herein as “CXCL12-interacting heparinoids”.

In some embodiments, the CXCL12-interacting heparinoid inhibits bindingof CXCL12 to CXCR4 with an IC₅₀ of about 0.05 μg/ml or less, about 0.04μg/ml or less, about 0.03 μg/ml or less, about 0.02 μg/ml or less, orabout 0.01 μg/ml or less in the assay set forth in Example 3. In someembodiments, the CXCL12-interacting heparinoid inhibits binding ofCXCL12 to CXCR4 with an IC90 of about 0.7 μg/ml or less, about 0.6 μg/mlor less, about 0.5 μg/ml or less, or about 0.4 μg/ml or less in theassay set forth in Example 3. In some embodiments, theCXCL12-interacting heparinoid is characterized by an IC50 of about 0.01μg/ml and an IC90 of about 0.5 μg/ml as determined by the method inExample 3. In some embodiments, the CXCL12-interacting heparinoid iscapable of inhibiting CXLC12/CXCR4 interaction, as measured by themethod set forth in Example 3, which is about the same as an equivalentweight of unfractionated heparin.

In typical embodiments, the CXCL12-interacting heparinoid is capable ofeffecting at least 20% inhibition of the binding of CXCL12 to CXCR4 inthe assay set forth in Example 3 at a concentration that, if achieved inplasma, would not effect substantial anticoagulation. In variousembodiments, the CXCL12-interacting heparinoid is capable of effectingat least 25% inhibition of the binding of CXCL12 to CXCR4 in the assayset forth in Example 3 at a concentration that, if achieved in plasma,would not effect substantial anticoagulation. In certain embodiments,the CXCL12-interacting heparinoid is capable of effecting at least 30%,at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, atleast 60% inhibition of the binding of CXCL12 to CXCR4 in the assay setforth in Example 3 at a concentration that, if achieved in plasma, wouldnot effect substantial anticoagulation. In specific embodiments, theCXCL12-interacting heparinoid is capable of effecting at least 65%, atleast 70%, at least 80%, at least 85%, even at least 90%, 91%, 92%, 93%,94%, 95% inhibition of the binding of CXCL12 to CXCR4 in the assay setforth in Example 3 at a concentration that, if achieved in plasma, wouldnot effect substantial anticoagulation. In particular embodiments, theCXCL12-interacting heparinoid is capable of effecting at least 96%, 97%even at least 98% inhibition of the binding of CXCL12 to CXCR4 in theassay set forth in Example 3 at a concentration that, if achieved inplasma, would not effect substantial anticoagulation.

In various embodiments, the CXCL12-interacting heparinoid is capable ofbinding to CXCL12 under physiological conditions.

In preferred embodiments, the CXCL12-interacting heparinoid is aderivative of USP heparin (also known as “unfractionated heparin”,“UFH”) that is substantially desulfated at the 2-O position ofα-L-iduronic acid (referred to herein as the “2-O position”) and/or 3-Oposition of D-glucosamine-N-sulfate (6-sulfate) (referred to herein asthe “3-O position”). In preferred embodiments, the 2-O, 3-O-desulfatedheparin derivative is not substantially desulfated at the 6-O or Npositions.

For purposes of the present disclosure, the percentage desulfation atthe 2-O position of a sample of 2-O, 3-O-desulfated heparin derivative(“ODSH”) is defined as the percentage reduction in sulfate functionalgroups on the 2-O position of the 2-O-sulfo-α-L-iduronic acid residuesas compared to the sulfate functional groups on the 2-O positions of the2-O-sulfo-α-L-iduronic acid residues in a sample of the 6thInternational Standard for Unfractionated Heparin, NIBSC code 07/328(“NIBSC standard”). For purposes of the present disclosure, thepercentage desulfation at the 3-O position of a sample of ODSH isdefined as the percentage reduction in sulfate functional groups on the3-O position of the2-deoxy-2-sulfamido-3-O-sulfo-α-D-glucopyranosyl-6-O-sulfate residues ascompared to the sulfate functional groups on the 3-O positions of the2-deoxy-2-sulfamido-3-O-sulfo-α-D-glucopyranosyl-6-O-sulfate residues ina sample of the NIB SC standard.

In some embodiments, the CXCL12-interacting heparinoid is at least 85%,at least 90%, at least 95%, or at least 99% desulfated at the 2-Oposition. In some embodiments, the CXCL12-interacting heparinoids are atleast 85%, at least 90%, at least 95%, or at least 99% desulfated at the3-O position. In some embodiments, the CXCL12-interacting heparinoidsare at least 85%, at least 90%, at least 95%, at least 99% desulfated atthe 2-O position and the 3-O position.

For purposes herein, average molecular weight of heparinoids isweight-average molecular weight, Mw, and is determined by size exclusionchromatography according to the USP monograph for Enoxaparin sodium,with USP Heparin MW Calibrant used as an additional calibrant.

In some embodiments, the CXCL12-interacting heparinoids have an averagemolecular weight from about 2 kDa to about 15 kDa. In some embodiments,the CXCL12-interacting heparinoids have an average molecular weight ofat least about 2 kDa, at least about 3 kDa, at least about 4 kDa, atleast about 5 kDa, at least about 6 kDa, or at least about 7 kDa. Insome embodiments, the CXCL12-interacting heparinoids have an averagemolecular weight of less than about 15 kDa, less than about 14 kDa, lessthan about 13 kDa, less than about 12 kDa, less than about 11 kDa, lessthan about 10 kDa, or less than about 9 kDa. In some embodiments, theaverage molecular weight of the CXCL12-interacting heparinoid isselected from about 2 kDa, 3 kDa, 4 kDa, 5 kDa, 6 kDa, 7 kDa, 8 kDa, 9kDa, 10 kDa, 11 kDa, 12 kDa, 13 kDa, 14 kDa, 15 kDa, 16 kDa, 17 kDa, 18kDa, or a range that includes any of these values as endpoints.

In some embodiments, the substantially 2-O, 3-O desulfatedCXCL12-interacting heparinoid for use in the methods described hereinare compositions in which the average molecular weight is at least about8 kDa. In some embodiments, the substantially 2-O, 3-O desulfatedCXCL12-interacting heparinoids have an average molecular weight ofgreater than about 8 kDa. In various embodiments, the substantially 2-O,3-O desulfated CXCL12-interacting heparinoids have an average molecularweight ranging from about 8 kDa to about 15 kDa. In some embodiments,the substantially 2-O, 3-O desulfated CXCL12-interacting heparinoids foruse in the methods described herein have an average molecular weightthat ranges in size from about 11 kDa to about 13 kDa.

An exemplary CXCL12-interacting heparinoid is substantially 2-O, 3-Odesulfated heparin, referred to herein as ODSH. ODSH for use in theabove-described methods can be prepared from bovine or porcine heparin.In an exemplary method of preparing ODSH from porcine heparin, ODSH issynthesized by cold alkaline hydrolysis of USP porcine intestinalheparin, which removes the 2-O and 3-O sulfates, leaving N- and 6-Osulfates on D-glucosamine sugars and carboxylates on α-L-iduronic acidsugars substantially intact (Fryer et al., J. Pharmacol. Exp. Ther. 282:208-219 (1997), incorporated herein by reference in its entirety). Usingthis method, ODSH can be produced with an average molecular weight ofabout 11.7±0.3 kDa. Additional methods for the preparation ofsubstantially 2-O, 3-O desulfated CXCL12-interacting heparinoids mayalso be found, for example, in U.S. Pat. Nos. 5,668,118, 5,912,237, and6,489,311, and WO 2009/015183, the contents of which are incorporatedherein in their entirety, and in U.S. Pat. Nos. 5,296,471; 5,969,100;and 5,808,021.

In contrast to unfractionated heparin, ODSH is substantiallynon-anticoagulating: administered to a subject at a dose that isequivalent in weight to a fully-anticoagulating dose of unfractionatedheparin, the clotting time measured in an aPTT assay is no greater than45 seconds, and typically in the upper range of normal, where normalclotting time ranges from about 27 to 35 seconds. By comparison,unfractionated heparin administered to a subject at a fullyanticoagulant dose causes time to clot to range from about 60 to about85 seconds in an aPTT assay.

Thus, in certain preferred embodiments, the CXCL12-interactingheparinoid is substantially non-anticoagulating. In preferredembodiments, the CXCL12-interacting heparinoid, if administered to asubject at a dose that is weight equivalent to a fully-anticoagulatingdose of unfractionated heparin, the clotting time measured in an aPTTassay is no greater than 45 seconds.

Another measure of ODSH's anticoagulant activity is its anti-X_(a)activity which can be determined in an assay carried out using plasmatreated with Russell viper venom. In specific examples, ODSH exhibitedless than 9 U of anticoagulant activity/mg in the USP anticoagulantassay (e.g., 7±0.3 U), less than 5 U of anti-Xa activity/mg (e.g.,1.9±0.1 U/mg) and less than 2 U of anti-II_(a) activity/mg (e.g.,1.2±0.1 U/mg) (compared to unfractionated heparin which has an activityof 165-190 U/mg in all three assays; Rao et al., Am. J. Physiol.299:C97-C110 (2010), incorporated herein by reference in its entirety).Thus, in certain embodiments, the CXCL12-interacting heparinoid exhibitsless than 9 U of anticoagulant activity/mg in the USP anticoagulantassay, and/or less than 5 U of anti-X_(a) activity/mg, and/or less than2 U of anti-IIa activity/mg.

Furthermore, ODSH has a low affinity for anti-thrombin III (Kd˜339 μM or4 mg/ml vs. 1.56 μM or 22 μg/ml for unfractionated heparin), consistentwith the observed low level of anticoagulant activity, measured asdescribed in Rao et al., supra, at page C98. Thus, in certainembodiments, the CXCL12-interacting heparinoid has a low affinity foranti-thrombin III (Kd˜339 μM or 4 mg/ml).

In some embodiments, the CXCL12-interacting heparinoids have no morethan 40% of the anticoagulating activity of an equal weight ofunfractionated heparin by any one or more of the above-described tests.In some embodiments, the CXCL12-interacting heparinoid has no more than35%, no more than 30%, no more than 20%, no more than 10%, no more than9%, no more than 8%, no more than 7%, no more than 6%, no more than 5%,no more than 4%, no more than 3%, no more than 2%, or no more than 1% ofthe anti-coagulating activity of an equal weight of unfractionatedheparin by any one or more of the above-described tests.

In some embodiments, the CXCL12-interacting heparinoid does not triggerplatelet activation and does not induce heparin-induced thrombocytopenia(HIT). Platelet activation can be determined using a serotonin releaseassay, for example as described in U.S. Pat. No. 7,468,358 and Sheridanet al., Blood 67:27-30 (1986), incorporated herein by reference. In someembodiments, the CXCL12-interacting heparinoid is capable of bindingplatelet factor 4, also referred to as chemokine (C-X-C motif) ligand 4(CXCL4).

In some embodiments, the CXCL12-interacting heparinoid is a lowmolecular weight heparin (LMWH). “Low molecular weight heparin” or“LMWH” refers to heparin fragments that have a mean molecular weight ofabout 4 to about 6 kDa. In some embodiments, the LMWHs have a molecularweight distribution of about 1000 to about 10000. LMWHs are typicallymade by chemical or enzymatic depolymerization of heparin, generallyunfractionated heparin, and can be further purified to select theappropriate size of the LMWH. The LMWH can be prepared using a number ofdifferent separation or fractionation techniques known to and used bythose of skill in the art, including, for example, gel permeationchromatography (GPC), high-performance liquid chromatography (HPLC),ultrafiltration, size exclusion chromatography, and the like.

In certain embodiments, the LMWH is selected from the group consistingof bemiparin, nadroparin, reviparin, enoxaparin, parnaparin, certoparin,dalteparin, tinzaparin, and necuparanib.

In typical embodiments, the CXCL12-interacting heparinoid displays bonemarrow cell mobilizing activity, particularly HSC mobilizing activity,more particularly bone marrow-residing cancer cell mobilizing activity.In some embodiments, the CXCL12-interacting heparinoid is characterizedby about 50% or more, 60% or more, 70% or more, 80% or more, 90% ormore, or 95% or more of the HSC mobilizing activity of an equivalentweight of unfractionated heparin. HSC mobilizing activity can bemeasured by mobilization of cells having one or more of the markerprofiles listed in Table 1 below. In certain embodiments, HSC mobilizingactivity is measured using at least the CD34⁺ marker phenotype.

TABLE 1 CD34⁺ CD34⁺ CD38⁻ CD34⁺ Lin⁻ Thy1⁺ CD34⁺ c-kit⁺ CD34⁺ Tie⁺ CD34⁺CD133⁺ CD34⁻ Lin⁻ CD133⁻ CD7⁻ CD34⁺ CD38⁻ Lin⁻ Rhodamine123^(low) CD34⁺CD38⁻ Lin⁻ CD45RA⁻ Rhodamine123^(low) CD49f⁺

5.2.4. Administration of CXCL12-Interacting Heparinoid

In one aspect, the methods described herein comprise administering to asubject receiving an antineoplastic treatment regimen aCXCL12-interacting heparinoid, wherein the cancer is one that isprivileged by CXCL12-CXCR4 interaction against therapeutic intervention.In certain embodiments, the cancer is one in which neoplastic cells,such as cancer stem cells, migrate to and/or reside in privilegedanatomic sites. In some embodiments, the cancer is characterized bystromal expression of CXCL12. In embodiments, the CXCL12-interactingheparinoid is administered in an amount and at a time effective toenhance effectiveness of the antineoplastic treatment regimen.

In another aspect, the methods comprise administering to a subjectreceiving a treatment regimen for a hematopoietic stem cell disorder aheparin derivative capable of inhibiting binding of CXCL12 to CXCR4. Theheparin derivative is administered in an amount and at a time effectiveto enhance effectiveness of the treatment regimen.

5.2.4.1. Routes of Administration

The CXCL12-interacting heparinoid can be administered in the methodsdescribed herein by any one or more of a variety of routes.

In certain embodiments, the CXCL12-interacting heparinoid isadministered intravenously. In certain embodiments, theCXCL12-interacting heparinoid is administered by bolus intravenousadministration. In some embodiments, a bolus dose is administered overless than a minute, about a minute, about 2 minutes, about 3 minutes,about 4 minutes, or about 5 minutes. In some embodiments, theCXCL12-interacting heparinoid is administered by continuous intravenousinfusion. In other embodiments, the CXCL12-interacting heparinoid isadministered by subcutaneous injection. In some embodiments, theCXCL12-interacting heparinoid is administered as one or more bolusintravenous injections preceded and/or followed by continuous infusion.

5.2.4.2. Effective Amounts

The CXCL12-interacting heparinoid is administered in an amount effectiveto enhance efficacy of the treatment regimen. In methods of treatingcancers, the CXCL12-interacting heparinoid is administered in an amounteffective to enhance the efficacy of the antineoplastic treatmentregimen. In methods of treating hematopoietic stem cell disorders, theCXCL12-interacting heparinoid is administered in an amount effective toenhance the efficacy of the treatment regimen used to treat thedisordered HSC cells.

In some embodiments, the enhancement of treatment efficacy is withrespect to one or more of the anti-tumor effect, the response rate(e.g., overall or objective response rate), the time to diseaseprogression or the survival rate (e.g., progression free survival oroverall survival). Anti-tumor effects include, but are not limited to,inhibition of tumor growth, tumor growth delay, regression of tumor,shrinkage of tumor, increased time to regrowth of tumor on cessation oftreatment, and/or slowing of disease progression.

In typical embodiments, the CXCL12-interacting heparinoid isadministered in an amount effective to mobilize neoplastic cells from aprivileged anatomic site. Typically, the amount is effective to mobilizeneoplastic cells from the bone marrow.

In some embodiments, the CXCL12-interacting heparinoid is administeredin an amount effective to increase the number of cancer cells outsidethe bone marrow, e.g., in the peripheral blood and/or peripheraltissues. In some embodiments, the CXCL12-interacting heparinoid isadministered in an amount effective to decrease the number of cancercells in the bone marrow.

In preferred embodiments, the CXCL12-interacting heparinoid isadministered in an amount effective to decrease the number of cancercells in the bone marrow by at least 50%. In certain embodiments, theCXCL12-interacting heparinoid is administered in an amount effective todecrease the number of cancer cells in the bone marrow by at least 60%,at least 70%, at least 80%, or more. In specific embodiments, theCXCL12-interacting heparinoid is administered in an amount effective todecrease the number of cancer cells in the bone marrow by at least 85%,at least 90%, even by as much as 95% or more.

In certain embodiments, the diminution of cancer cells in the bonemarrow is measured by visual inspection of bone marrow biopsies.

In some embodiments, the mobilization of bone-marrow residing cancercells to the peripheral blood or tissues, and/or the decrease in thenumber of cancer cells in the bone marrow, is determined by detectingand quantifying a cancer cell marker or a set of cell markersdistinctive for or indicative of the cancer cell.

In some embodiments, the cancer cell marker can include one or more of acell surface marker, a cellular enzyme, a cellular genotype, andcombinations thereof. By way of example and not limitation, markersuseful for assessing mobilization of the relevant cancer cells are givenbelow.

TABLE 2 Cancer Type Markers Lung cancer Adrenocorticotropic Hormone(ACTH) Calcitonin EGFR mutation Breast cancer Cancer Antigen 15-3 CancerAntigen 549 C-erb B-2 Prostate Cancer Acid Phosphatase Prostate SpecificAntigen Carcinoembryonic antigen Kidney Cancer PAX-2 Renal cellcarcinoma marker antigen (RCCM) Kidney-specific cadherin (KSC)Neuroblastoma Cyclin D1 GALNT13 GD2 disialoganglioside AcuteLymphoblastic Neprilysin (CALLA antigen) Leukemia TEL-AML1 fusion AcuteMyeloid Leukemia NPM1 mutations FLT3 mutations CMBPA mutations ChronicLymphocytic CD38 leukemia zeta-associated protein (ZAP)-70 IgVHmutations Chronic Myelogenous Philadelphia Chromosome (Ph1: bcr-ablfusion) Leukemia Acute Monocytic CD13 Leukemia CD33 CD11b, CD11c

Methods for detecting the cell markers and cancer cells include, amongothers, flow cytometry (e.g., fluorescence activated cell sorting);immune detection (e.g., histochemistry); polymerase chain reaction (andother methods for detecting gene polymorphisms); fluorescence in situhybridization; gene expression profiling; proteomics; morphologicalanalysis; and combinations thereof. The sensitivity of each of thedetection techniques can vary, and the appropriate method selected basedon sensitivity appropriate for the treatment. For example, for acutemyeloid leukemia (AML), detection sensitivity—the number of blast cellsthat can be detected per 100,000 cells—of standard detection approachesis as follows: morphological detection with immunohistochemistry candetect from about 1000 to about 5000 blast cells per 100,000 cells;karyotype analysis can detect about 5000 blast cells per 100,000 cells;flow cytometry can detect about 10 blast cells per 100,000 cells; andpolymerase chain reaction can detect about 0.1 blasts per 100,000 cells.In some embodiments, the bone is imaged, for example with a MRI scan, aCT scan, and/or a PET scan to detect presence of cancer in the bonemarrow, metastasis of cancers into the bone marrow, and/or any changesarising from administration of the CXCL12-interacting heparinoid.

In some embodiments, the CXCL12-interacting heparinoid is administeredas an intravenous bolus. In certain embodiments, the CXCL12-interactingheparinoid is administered in an intravenous bolus of no more than about1 mg/kg patient body weight. In typical intravenous bolus dosingembodiments, the CXCL12-interating heparinoid is administered at a doseof no more than about 25 mg/kg. In various embodiments, theCXCL12-interacting heparinoid is administered at an intravenous bolusdose of at least about 2 mg/kg, at least about 3 mg/kg, at least about 4mg/kg, at least about 5 mg/kg, at least about 6 mg/kg, at least about 7mg/kg, at least about 8 mg/kg, at least about 9 mg/kg, even at leastabout 10 mg/kg. In some embodiments, the bolus is at least about 15mg/kg, even at least about 20 mg/kg. In certain preferred embodiments,the bolus is about 4 mg/kg. In certain other preferred embodiments, thebolus is about 8 mg/kg. In certain preferred embodiments, the bolus isabout 20 mg/kg.

In some embodiments, the CXCL12-interacting heparinoid is administeredin a bolus of from about 2 to about 25 mg/kg, from about 2 mg/kg toabout 20 mg/kg, from about 2 mg/kg to about 15 mg/kg, from about 3 mg/kgto about 10 mg/kg, or from about 4 mg/kg to about 8 mg/kg.

In some embodiments, the CXCL12-interacting heparinoid is administeredas an intravenous infusion. In certain embodiments, the infusion is at adose rate of at least about 0.1 mg/kg/hr, at least about 0.2 mg/kg/hr,at least about 0.3 mg/kg/hr, at least about 0.4 mg/kg/hr, at least about0.5 mg/kg/hr, at least about 1 mg/kg/hr, even at least about 2 mg/kg/hr.In various embodiments, the CXCL12-interacting heparinoid isadministered at an infusion rate of no more than about 5 mg/kg/hr. Incertain embodiments, the CXCL12-interacting heparinoid is administeredat an infusion rate of no more than about 4 mg/kg/hr, 3 mg/kg/hr, about2 mg/kg/hr, even no more than about 1 mg/kg/hr.

In typical embodiments, infusions at the above-described dose rates areadministered continuously for up to 7 days. In certain embodimentsinfusions at the above-described dose rates are administeredcontinuously for up to 6 days, 5 days, 4 days, or 3 days. In someembodiments, infusions at the above-described dose rates areadministered continuously for up to 2 days or up to 24 hours. In someembodiments, infusions at the above-described rates are administered forup to 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 12 hours, 16 hours,or up to 24 hours or more. In certain embodiments, the infusions at theabove described dose rates are administered for the duration of eachcycle of treatment.

In some embodiments, the CXCL12-interacting heparinoid is administeredas an initial bolus of about 20 mg/kg, optionally followed by aninfusion of up to about 2 mg/kg/hour for up to about 4 hours, 8 hours,12 hrs, 16 hours, even up to about 24 hours. In one embodiment, theCXCL12-interacting heparinoid is administered as an initial bolus ofabout 8 mg/kg, optionally followed by an infusion of about 0.5mg/kg/hour for at least about 8 hours. In some embodiments, theCXCL12-interacting heparinoid is administered as an intravenous bolus ata dose of about 4 mg/kg, optionally followed by an intravenous infusionof the CXCL12-interacting heparinoid at a dose of about 0.25mg/kg/hr—about 0.375 mg/kg/hr for at least 24 hours. In someembodiments, the CXCL12-interacting heparinoid is administered as anintravenous bolus at a dose of about 4 mg/kg, followed by a continuousintravenous infusion at a rate of 0.25 mg/kg/hr for a total of 7 days.In some embodiments, the CXCL12-interacting heparinoid is administeredas a 4 mg/kg bolus on Day 1 followed by a continuous intravenousinfusion of 0.25 mg/kg/hr for days 1 through 5 of each 28-day cycle.

For subcutaneous administration, CXCL12-interacting heparinoid can beadministered at doses ranging from about 25 mg to about 400 mg, about 50mg to about 300 mg, or about 75 mg to about 200 mg, in volumes of 2.0 mLor less per injection. In some embodiments, the CXCL12-interactingheparinoid at the above-described dosages are administeredsubcutaneously each day for up to 7 days. In certain embodiments, theCXCL12-interacting heparinoid at the above-described dosages areadministered subcutaneously each day for up to 6 days, 5 days, 4 days,or 3 days. In some embodiments, CXCL12-interacting heparinoid at theabove-described dosages are administered subcutaneously for up to 2 daysor up to 24 hours. In some embodiments, CXCL12-interacting heparinoid atthe above-described dosages are administered subcutaneously each day forthe duration of the cycle of treatment.

5.2.4.3. Effective Timings

In various embodiments, the CXCL12-interacting heparinoid isadministered adjunctively with the antineoplastic treatment regimen. Theterms “adjunctive administration”, “adjunctively administering” or“administering adjunctive to” are used interchangeably herein to meanadministering the CXCL12-interacting heparinoid in therapeuticallyeffective temporal proximity to the antineoplastic treatment regimen,that is, in sufficient temporal proximity to administration of theantineoplastic treatment regimen as to enhance the efficacy of theantineoplastic treatment regimen. In some embodiments, theCXCL12-interacting heparinoid is administered prior to treatment withthe antineoplastic treatment. In some embodiments, theCXCL12-interacting heparinoid is administered concurrently withtreatment with the antineoplastic treatment regimen. In someembodiments, the CXCL12-interacting heparinoid is administered prior toand concurrently with the antineoplastic treatment regimen.

In the methods described herein, the antineoplastic treatment regimencan involve one or more of an induction therapy, one or more of aconsolidation therapy, and/or one or more of a maintenance therapy. Insome embodiments, the consolidation or maintenance therapy can beoptional. For example, a treatment regimen can include an inductiontherapy followed by maintenance therapy, or an induction therapyfollowed by consolidation therapy without any maintenance therapy. It isalso to be understood that each of induction therapy, consolidationtherapy, and maintenance therapy can have one or more cycles oftreatment. As such, in some embodiments, the induction therapy can haveone or more cycles of induction treatment; the consolidation therapy canhave one or more cycles of consolidation treatment; and the maintenancetherapy can have one or more cycles of maintenance treatment.

Thus, in various embodiments, the CXCL12-interacting heparinoid isadministered adjunctively to one or more cycles of induction therapy. Incertain embodiments, the CXCL12-interacting heparinoid is administeredadjunctively to one or more cycles of consolidation therapy. In someembodiments, the CXCL12-interacting heparinoid is administeredadjunctively to one or more cycles of maintenance therapy.

In some embodiments, the CXCL12-interacting heparinoid is administeredat a time sufficiently prior to treatment with the antineoplastictreatment regimen as to mobilize the cancer cells from the privilegedanatomic site, such as the bone marrow, before administration of theantineoplastic agent(s). In some embodiments, the CXCL12-interactingheparinoid is administered at least about 1 hr to about 24 hr prior totreatment with the antineoplastic therapeutic agent. In someembodiments, the CXCL12-interacting heparinoid is administered at least2 days or more, or 3 days or more prior to treatment with theantineoplastic therapeutic. In certain embodiments, theCXCL12-interacting heparinoid is administered both prior to andconcurrently with the antineoplastic treatment regimen.

In some embodiments, the CXCL12-interacting heparinoid is administeredprior to induction therapy, particularly prior to each cycle ofinduction therapy with an antineoplastic therapeutic. In someembodiments, the CXCL12-interacting heparinoid is administered at a highdose prior to the induction therapy, particularly prior to each cycle ofinduction therapy. In some embodiments, the CXCL12-interactingheparinoid is administered prior to, during and optionally, following,treatment with the antineoplastic therapeutic used in the inductiontherapy, such as by continuous administration, for example to keepcancer cells from reestablishing residence in the bone marrow or otherprivileged anatomic site.

In some embodiments, the CXCL12-interacting heparinoid is administeredprior to consolidation therapy, particularly prior to each cycle ofconsolidation therapy with an antineoplastic therapeutic agent. In someembodiments, the CXCL12-interacting heparinoid is administered at a highto moderate dose prior to the consolidation therapy, particularly priorto each cycle of consolidation therapy. In some embodiments, theCXCL12-interacting heparinoid is administered prior to, during andoptionally, following, treatment with the antineoplastic therapeuticused in the consolidation therapy, such as by continuous administration,for example to keep cancer cells from reestablishing residence in thebone marrow or other privileged anatomic sites.

In some embodiments, the CXCL12-interacting heparinoid is administeredprior to maintenance therapy, particularly prior to each cycle ofmaintenance therapy. In some embodiments, the CXCL12-interactingheparinoid is administered at a high to moderate dose, particularly at amoderate dose, prior to maintenance therapy, particularly prior to eachcycle of maintenance therapy. In some embodiments, theCXCL12-interacting heparinoid is administered prior to, during, andoptionally following treatment with the antineoplastic therapeutic inthe maintenance therapy used in the maintenance therapy, such as bycontinuous administration, for example to keep cancer cells fromreestablishing residence in the bone marrow or other privileged anatomicsites.

In some embodiments, the CXCL12-interacting heparinoid is administeredas an adjunct to induction therapy, and is administered at high dose. Insome embodiments, the CXCL12-interacting heparinoid is administered asan adjunct to consolidation therapy, and is administered in a high doseto a moderate dose. In some embodiments, the CXCL12-interactingheparinoid is administered as an adjunct to maintenance therapy,particularly at a high to a moderate dose, more particularly a moderatedose of the CXCL12-interacting heparinoid.

In some embodiments, the subject is treated with a high dose of theCXCL12-interacting heparinoid prior to induction therapy with theantineoplastic therapeutic, followed by treatment with a high tomoderate dose of the CXCL12-interacting heparinoid for each cycle of aconsolidation and/or maintenance therapy with the antineoplastictherapeutic. In some embodiments, the subject is treated with a highdose of the CXCL12-interacting heparinoid prior to induction therapywith the antineoplastic therapeutic, followed by treatment with a highto moderate dose of the CXCL12-interacting heparinoid for each cycle ofa consolidation therapy, and treatment with a high to moderate dose,particularly a moderate dose of the CXCL12-interacting heparinoid foreach cycle of a maintenance therapy. In each cycle of treatment, theCXCL12-interacting heparinoid can be administered as a bolus prior toadministration of the antineoplastic therapeutic. In some embodiments,the bolus administration can be followed by a continuous administration,particularly during and/or subsequent to treatment with theantineoplastic therapeutic.

As discussed above, in some embodiments, the CXCL12-interactingheparinoid is administered in coordination with the cycles of treatmentwith an antineoplastic therapeutic, particularly a non-myelosuppressiveantineoplastic therapeutic.

In some embodiments the CXCL12-interacting heparinoid is administered incoordination with hypomethylation agents. In certain embodiments thehypomethylation agent is azacitidine. In certain embodiments, theazacitidine is administered at 75 mg/m² as a 15 minute intravenousinfusion daily on days 1 through 5 of each 28-day cycle and a heparinoidis administered as a 4 mg/kg bolus on Day 1 followed by a continuousintravenous infusion of 0.25 mg/kg/hr for days 1 through 5 of each28-day cycle.

An exemplary treatment protocol follows the following schedule:treatment cycle length: every 21 days for 4 cycles. On days 1 to 3 ofeach cycle, the subject is treated with an antineoplastic therapeutic,such as a non-myelosuppressive therapeutic. ODSH is administered as an 8hour infusion, on days 1-5, of weeks 1, 2 and 3 and then on days 1-3 ofsubsequent cycles as an 8 hour infusion. The dose is about 8 mg/kg bolusfollowed by about 0.5 mg/kg/hour infusion. In some embodiments, the doseis about 20 mg/kg bolus followed by about 2 mg/kg/hour infusion, such asduring the induction therapy.

5.2.4.4. Duration and Frequency of Administration

In typical embodiments, the CXCL12-interacting heparinoid isadministered for up to 1 hour. In various embodiments, theCXCL12-interacting heparinoid is administered for up to 4 hours. Incertain embodiments, the CXCL12-interacting heparinoid is administeredfor up to 6 hours, even up to 8 hours. In some embodiments, theCXCL12-interacting heparinoid is administered for up to 12 hours, 18hours, even up to 24 hours. In certain embodiments, theCXCL12-interacting heparinoid is administered for up to 2 days, 3 days,4 days, 5 days, 6 days, or a week or more. The CXCL12-interactingheparinoid can be administered, in some embodiments, for periods of morethan a week, including 1 month, 2 months, 3 months or more.

Typically, CXCL12-interacting heparinoid administration is repeated. Forexample, in certain embodiments, heparinoid is administered once daily,twice daily, three times daily, four times daily, five times daily,every two days, every three days, every five days, once a week, onceevery two weeks, once a month, every other month, semi-annually, orannually. In some embodiments, the CXCL12-interacting heparinoid isadministered at regular intervals over a period of several weeks,followed by a period of rest, during which no heparinoid isadministered. For example, in some embodiments, CXCL12-interactingheparinoid is administered for one, two, three, or more weeks, followedby one, two, three, or more weeks without heparinoid administration. Therepeated administration can be at the same dose or at a different dose.The CXCL12-interacting heparinoid can be administered in one or morebolus injections, one or more infusions, or one or more bolus injectionsfollowed or preceded by infusion.

The frequency of dosing can be based on and adjusted for thepharmacokinetic parameters of the CXCL12-interacting heparinoid and theroute of administration. Dosages are adjusted to provide sufficientlevels of the CXCL12-interacting heparinoid or to maintain the desiredphysiological effect, particularly a therapeutic effect. Any effectiveadministration regimen regulating the timing and sequence of doses maybe used, as discussed herein.

Accordingly, the pharmaceutical compositions can be administered in asingle dose, multiple discrete doses, continuous infusion, sustainedrelease depots, or combinations thereof, as required to maintain desiredminimum level of the agent. Daily dosages may vary, depending on thespecific activity of the particular heparinoid. Depending on the routeof administration, a suitable dose may be calculated according to, amongothers, body weight, body surface area, or organ size. The final dosageregimen will be determined by the attending physician in view of goodmedical practice, considering various factors that modify the action ofdrugs, e.g., the agent's specific activity, the severity of the diseasestate, the responsiveness of the patient, the age, condition, bodyweight, sex, and the like. Additional factors that may be taken intoaccount include time and frequency of administration, drugcombination(s), reaction sensitivities, and tolerance/response totherapy. Further refinement of the dosage appropriate for treatmentinvolving any of the formulations mentioned herein is done by theskilled practitioner, especially in light of the dosage information andassays disclosed, as well as the pharmacokinetic data observed inclinical trials. The amount and/or frequency of the dosage can bealtered, increased, or reduced, depending on the subject's response andin accordance with standard clinical practice. The proper dosage andtreatment regimen can be established by monitoring the progress oftherapy using conventional techniques known to skilled artisans.Appropriate dosages may be ascertained through use of established assaysfor determining concentration of the CXCL12-interacting heparinoid in abody fluid or other sample together with dose response data.

In embodiments in which CXCL12-interacting heparinoid is administered toa subject in combination with other therapeutic agents, theCXCL12-interacting heparinoid is administered in a therapeuticallyeffective temporal proximity to the treatment regimen with the othertherapeutic. Administration of a CXCL12-interacting heparinoid can beconcurrent with (at the same time), sequential to (at a different timebut on the same day, e.g., during the same patient visit), or separatefrom (on a different day) the treatment with the other therapeutic. Insome embodiments, the CXCL12-interacting heparinoid is administeredconcurrently, sequentially, and/or separately from the other agent ortherapy being administered. When administered sequentially orseparately, the CXCL12-interacting heparinoid can be administeredbefore, after, or both before and after the other treatment.

In embodiments in which the CXCL12-interacting heparinoid isadministered in combination with treatment with another therapeuticagent, the CXCL12-interacting heparinoid can be administrated via thesame or different route as the other therapeutic administered intemporal proximity. In some embodiments, the CXCL12-interactingheparinoid is administered concurrently or sequentially by the sameroute. For example, in some embodiments, the CXCL12-interactingheparinoid and the other therapeutic are administered intravenously,either concurrently or sequentially. Optionally, as part of a treatmentregimen, the CXCL12-interacting heparinoid can further be administeredseparately (on a different day) from the other therapeutic by adifferent route, e.g., subcutaneously. In some embodiments, theCXCL12-interacting heparinoid is administered intravenously on the sameday, either at the same time (concurrently), a different time(sequentially), or both concurrently and sequentially with the othertherapeutic, and is also administered subcutaneously on one or more dayswhen the patient is not receiving other treatment. In some embodiments,the CXCL12-interacting heparinoid is administered concurrently orsequentially by a different route. Optionally, as part of a treatmentregimen, the CXCL12-interacting heparinoid can further be administeredseparately (on a different day) from the other therapeutic by the sameor different route as that by which the other therapeutic isadministered.

Other methods for delivering CXCL12-interacting heparinoids in themethods presented herein can be adapted from those are described in U.S.Pat. No. 4,654,327, which describes oral administration of heparin inthe form of a complex with a quaternary ammonium ion; U.S. Pat. No.4,656,161, which describes a method for increasing the enteralabsorbability of heparinoids by orally administering the drug along witha non-ionic surfactant such as polyoxyethylene-20 cetyl ether,polyoxyethylene-20 stearate, other polyoxyethylene (polyethyleneglycol)-based surfactants, polyoxypropylene-1 5 stearyl ether, sucrosepalmitate stearate, or octyl-beta-D-glucopyranoside; U.S. Pat. No.4,703,042, which describes oral administration of a salt of polyanionicheparinic acid and a polycationic species; and U.S. Pat. No. 5,714,477,which describes a method for improving the bioavailability ofheparinoids by administering in combination with one or several glycerolesters of fatty acids.

5.2.5. Optional Steps

In some embodiments, the method of treatment further comprises the stepof measuring or determining the number of cancer cells mobilized bytreatment with the CXCL12-interacting heparinoid, particularly prior totreatment with the antineoplastic therapeutic. For example, ameasurement of the number of cancer cells can be taken prior toadministration of the CXCL12-interacting heparinoid and subsequent toadministration of the CXCL12-interacting heparinoid to determine theincrease in the number of cancer cell mobilized by theCXCL12-interacting heparinoid treatment. However, it is to be understoodthat, in some embodiments, the treatments herein can be given beforemetastasis or even when no increase in peripheral cancer cells aremeasured, particularly given that in some cancers a reservoir of cancercells can remain in the bone marrow at levels not readily detectable,for example where there is minimal residual disease. Moreover,mobilization per se need not be a requisite condition for treatmentbecause the CXCL12-interacting heparinoid may dislodge the cancer cellssufficiently to increase susceptibility to the antineoplastictherapeutic without inducing movement of cancer cells to the peripheralblood or tissues.

5.3. Pharmaceutical Compositions and Unit Dosage Forms ofCXCL12-Interacting Heparinoids

In the methods presented herein, the CXCL12-interacting heparinoid isadministered in the form of a pharmaceutical composition.

In typical embodiments, the pharmaceutical composition comprises theCXCL12-interacting heparinoid and a pharmaceutically acceptable carrier,excipient, and/or diluent, and is formulated for parenteraladministration.

5.3.1. Pharmaceutical Compositions Formulated for I.V. Administration

In certain embodiments, pharmaceutical compositions of theCXCL12-interacting heparinoid are formulated in volumes andconcentrations suitable for intravenous administration. In someembodiments, the composition is formulated for bolus administration. Incertain embodiments, pharmaceutical compositions of theCXCL12-interacting heparinoid are formulated in volumes andconcentrations suitable for intravenous infusion.

Typical embodiments formulated for intravenous administration comprisethe CXCL12-interacting heparinoid in concentrations of at least about 10mg/ml. In various embodiments, the CXCL12-interacting heparinoid ispresent in a concentration of at least about 15 mg/ml, at least about 20mg/ml, at least about 30 mg/ml, at least about 40 mg/ml, at least about50 mg/ml. In certain embodiments, the CXCL12-interacting heparinoid ispackaged in sterile-filled 10 ml glass vials containing an isotonic 50mg/ml solution of heparinoid in buffered saline.

5.3.2. Pharmaceutical Compositions Formulated for S.C. Administration

In various embodiments, the pharmaceutical composition is formulated forsubcutaneous administration.

In certain such embodiments, the CXCL12-interacting heparinoid isassociated with multivalent cations. The term “associated”, when used todescribe the relationship between a heparinoid and a cation, means achemically relevant association. The association may be as a salt,ion/counterion, complex, binding, coordination or any other chemicallyrelevant association. The exact nature of the association will bereadily apparent to a person of skill in the art depending on the formof the composition.

In various such embodiments, the multivalent cations are selected fromcations having a charge of +2, +3, +4, or greater. In some embodiments,the multivalent cation is an ion that contains both positive andnegative charges, with a net charge greater than +1. Exemplarymultivalent cations include metal ions, amino acids, and other organicand inorganic cations. In certain embodiments, the ion is a metal ionthat is Zn²⁺, Ca²⁺, Mg²⁺ or Fe²⁺. In a specific embodiment, the cationis Ca²⁺. In another specific embodiment, the cation is Mg²⁺.

In certain of the embodiments of pharmaceutical composition intended forsubcutaneous administration, the CXCL12-interacting heparinoid isassociated primarily with one species of multivalent cation. In otherembodiments, the CXCL12-interacting heparinoid is associated withseveral different multivalent cation species. In specific embodiments,the CXCL12-interacting heparinoid is associated with Mg²⁺ and Ca²⁺.

In the multivalent cation embodiments, multivalent cations may beintroduced to the CXCL12-interacting heparinoid composition at any step.

In one embodiment, the CXCL12-interacting heparinoid is substantiallydesulfated at the 2-O and 3-O positions, and the multivalent cation ispresent during alkaline hydrolysis of the heparin starting material. Incertain embodiments, the multivalent cation is present as the chloridesalt. In certain embodiments, the multivalent cation is present as thehydroxide salt. In one embodiment, the chloride salt is preferred foruse during solution phase alkaline hydrolysis. In another embodiment,the hydroxide salt is preferred for use during solid phase alkalinehydrolysis. In another embodiment, the hydroxide salt is preferred foruse when alkaline hydrolysis is performed as a paste. Certainmultivalent cations may affect the level of desulfation if presentduring alkaline hydrolysis, and may be used to achieve desired levels ofdesulfation. The amount of the multivalent cation may be titrated tocontrol the amount of desulfation as described in U.S. Pat. no.5,296,471 at Example 4 therein.

Thus, when a multivalent cation is used during alkaline hydrolysis, themultivalent cation concentration used should be adjusted based on boththe desired level of desulfation and the desired concentration of thefinal product. The molar multivalent cation concentration used duringalkaline hydrolysis may be substantially less than the molar heparinconcentration. Preferably, the molar ratio (multivalent cation:heparin)is about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2,1.3, 1.4, or 1.5, or any ranges composed of those values. Preferably,the concentration of the multivalent cation used during alkalinehydrolysis is about 0.01 mM, 0.05 mM, 0.1 mM, 0.5 mM, 1 mM, 5 mM, 10 mM,20 mM, 50 mM, 100mM, 250 mM, 500 mM or 1M or any range composed of thosenumbers.

In certain embodiments, primarily monovalent cations are present duringthe cold alkaline hydrolysis step, and the multivalent cation is addedlater, during reconstitution of the lyophilate. In a most preferredembodiment, either MgCl₂ or CaCl₂ is added at high concentration duringreconstitution of the lyophilate.

The multivalent cation concentration used during reconstitution may beequal to the concentration of the cation used during alkalinehydrolysis. Preferably, the multivalent cation concentration is at leastabout 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold,10-fold, 15-fold, 20-fold, 25-fold, 30-fold, 50-fold, 75-fold, 100-fold,150-fold, 200-fold, 250-fold, 500-fold, or 1000-fold the concentrationof the cation used during alkaline hydrolysis. Preferably, theconcentration of the multivalent cation used during reconstitution isabout 0.1 M, 0.5 M, 1 M, 2 M, 3 M, 4 M, 5M, or greater. Most preferably,the concentration is about 2 M.

Excess cations can be removed by any method known to those in the art.One preferred method of removing excess cations is the use of adesalting column. Another preferred method of removing excess cations isdialysis. After removal of excess ions, the solution preferably hasabout equal, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold,9-fold, 10-fold, 15-fold, 20-fold, 25-fold, 30-fold, 50-fold, 75-fold,100-fold, 150-fold, 200-fold, 250-fold, 500-fold, or 1000-fold greatermultivalent cation concentration to monovalent cation concentration. Thesolution may also be free or substantially free of monovalent cations.

In typical embodiments, the final concentration of CXCL12-interactingheparinoid in the pharmaceutical composition is between 0.1 mg/mL and600 mg/mL. In certain embodiments, the final concentration of partiallydesulfated heparin in the pharmaceutical composition is between 200mg/mL and 400 mg/mL.

In some embodiments, the concentration of heparinoid is greater thanabout 25 mg/mL. In certain embodiments, the concentration of heparinoidis greater than about 50 mg/mL. In a variety of embodiments, theconcentration of heparinoid is greater than about 60 mg/mL, 70 mg/mL, 80mg/mL, 90 mg/mL, or 100 mg/mL.

In specific embodiments, the CXCL12-interacting heparinoid is present inthe pharmaceutical composition in a concentration greater than about 110mg/mL, 120 mg/mL, 130 mg/mL, 140 mg/mL, 150 mg/mL, 160 mg/mL, 170 mg/mL,180 mg/mL, or even greater than about 190 mg/mL or 200 mg/mL. Inspecific embodiments, the CXCL12-interacting heparinoid is present inthe pharmaceutical composition at a concentration of about 175 mg/mL. Inanother embodiment, the CXCL12-interacting heparinoid is present in thepharmaceutical composition at a concentration of about 200 mg/mL. In oneembodiment, the CXCL12-interacting heparinoid is present in thepharmaceutical composition at a concentration of 400 mg/mL.

In certain embodiments, the concentration of CXCL12-interactingheparinoid is 50 mg/mL to 500 mg/mL, 100 mg/mL to 400 mg/mL, or 150mg/mL to 300 mg/mL. In specific embodiments, the concentration is 50mg/mL, 100 mg/mL, 150 mg/mL, 200 mg/mL, 250 mg/mL, 300 mg/mL, 350 mg/mL,400 mg/mL, 450 mg/mL or 500 mg/mL. In certain currently preferredembodiments, the concentration is 200 mg/mL, 300 mg/mL or 400 mg/mL.

In typical embodiments, the pharmaceutical composition has a viscosityof less than about 100 cP. In various embodiments, the pharmaceuticalcomposition has a viscosity of less than about 80 cP. In certainembodiments, the pharmaceutical composition has a viscosity of less thanabout 60 cP. In particular embodiments, the pharmaceutical compositionhas a viscosity of less than about 20 cP.

In typical embodiments, the pharmaceutical composition has an osmolalityless than about 2500 mOsm/kg. In various embodiments, the pharmaceuticalcomposition has an osmolality between about 150 mOsm/kg and about 500mOsm/kg. In certain embodiments, the pharmaceutical composition has anosmolality between about 275 mOsm/kg and about 300 mOsm/kg. In aparticular embodiment, the pharmaceutical composition has an osmolalityof about 285 mOsm/kg. In a specific embodiment, the pharmaceuticalcomposition is isotonic.

6. EXAMPLES

Practice of the various embodiments of the treatment methods can beunderstood through reference to the following examples, which areprovided by way of illustration and are not intended to be limiting.

6.1. Example 1 CX-01 ODSH Attenuates Myelosuppressive Side Effects ofInduction Chemotherapy in Treatment of Acute Myeloid Leukemia, andSurprisingly Improves Remission Rate

A single arm open-label clinical study was conducted at multiple trialsites (University of Utah, Georgia Regents University, and MedicalUniversity of South Carolina) in patients with newly diagnosed acutemyeloid leukemia (AML) to confirm that ODSH could accelerate plateletand white blood cell (WBC) recovery in patients receiving inductionchemotherapy with a regimen known to have myelosuppressive side effects.

All patients received the following standard (“7+3”) induction regimen,

-   -   Idarubicin (12 mg/m²/day) by short intravenous infusion on Days        1, 2, and 3; and    -   Cytarabine (100 mg/m²) as a continuous intravenous infusion over        24 hours (Days 1 through 7).

All patients also received CX-01, a substantially 2-O, 3-O-desulfatedheparin derivative (ODSH) as an intravenous bolus immediately after theidarubicin dose on Day 1, at a dose of 4 mg/kg, followed by a continuousintravenous infusion at a dose of 0.25 mg/kg/hr for a total of 7 days(Days 1 through 7).

ODSH was manufactured under cGMP conditions by Scientific Protein Labs(Waunakee, Wis.) by cold alkaline hydrolysis of USP porcine intestinalunfractionated heparin during lyophilization. This process removes 2-Oand 3-O sulfates, leaving N-sulfates and 6-O sulfates and carboxylateslargely intact (Fryer et al., J. Pharmacol. Exp. Ther. 282:208-2219(1997)). Seven serial 1.2 kg batches of material have shown an averagemolecular weight of 11.7±0.3 kDa, low affinity for anti-thrombin III(Kd=339 μm, or 4 mg/ml) (vs. 1.56 μm or 22 μg/ml for UFH), andconsistently reduced USP anticoagulant activity (7±0.3 U ofanticoagulant activity/mg), anti-Xa activity (1.9±0.1 U/mg), andanti-IIa activity (1.2±0.1 U/mg) as compared with those of heparin(165-190 U/mg activity for all 3 assays). Drug product was formulated byPyramid Laboratories (Costa Mesa, Calif.) in sterile-filled 10 ml glassvials containing an isotonic 50 mg/ml solution of sodium ODSH inbuffered saline.

Twelve patients were enrolled. The median age was 56 (range 22-74).Based on cytogenetic, molecular, or antecedent hematologic disorder, 9of 12 patients fell into the intermediate or poor risk categories.Patients did not receive growth factor support, that is, Neupogen orsimilar agents, during induction cycles. Complete remission at the endof the induction cycle was assessed using International Working Group(IWG) criteria (see, e.g., Cheson et al., J. Clin. Oncol. 21:4642(2003)).

Platelet, neutrophil, and WBC recovery of the ODSH-treated AML patientswas compared with the recovery in historical control patients receivingidentical doses of idarubicin and cytarabine as induction therapy in aprevious clinical study comparing idarubicin and daunorubicin incombination with cytarabine (Vogler et al., J. Clin. Oncol.10(7):1103-11(1992)). In the previous study, 101 patients receivedidarubicin 12 mg/m² on days 1, 2, and 3 and cytarabine 100 mg/m² on days1-7. Growth factors were not administered to any patient.

Table 3 compares hematologic recovery parameters as reported in theprior study to those observed in the current study in which patientsadditionally received ODSH, as described above.

TABLE 3 Idarubicin + Idarubicin + Cytarabine Cytarabine + (n = 101) ODSH(n = 12) Time to platelet count >50,000 35 days 22 days Time toWBC >1000 31 days 21 days

In the current study, 11 out of 12 patients (92%), including twopatients who received an incomplete course of chemotherapy (3 and 5days, respectively), had a morphologic complete remission by IWGcriteria at the end of a single induction cycle. The only patient whodid not obtain a complete morphologic remission at the end of inductiontherapy presented with extensive mediastinal and peripherallymphadenopathy involved with granulocytic sarcomas, accompanying bonemarrow involvement with AML. This patient had residual extramedullarydisease at the end of his induction cycle, and achieved a completeremission with a subsequent cycle of FLAG-Ida chemotherapy without ODSH.

With 9 of 12 patients having intermediate or poor risk disease prior totreatment, and with two of these patients having received an incompletecourse of treatment, the 92% complete remission rate after the firstinduction cycle is higher than would otherwise be expected based onhistorical data, as shown in Table 4.

TABLE 4 Idarubicin + Idarubicin + Cytarabine Cytarabine + (n = 101) ODSH(n = 12) Complete response with 58% 92% first induction cycleFurthermore, 10 of the 12 patients remain in complete remission 5-13months after having been enrolled in the study.

FIG. 3 compares complete response rate of the 11 patients who enteredthe clinical trial with primary AML to historical controls, indicated byasterisk. The historical controls are 1,980 patients registered to 6studies conducted by the Eastern Cooperative Oncology Group (data fromRowe et al., Cancer 116(21):5012-5021 (2010).

Post-Induction Treatment and Outcomes

Of the 12 patients enrolled, 4 were not eligible to receivepost-induction treatment on study due to either age≥60 years, inductionfailure, or incomplete induction. Of the other patients, one developed aline-associated deep venous thrombosis requiring systemicanticoagulation and was taken off study before consolidation. Theremaining 4 patients each received one or more cycles of HIDAC and CX-01consolidation treatment on study as follows: Patient 1005 completed allfour cycles on study; Patient 1009 received 3 cycles of consolidation onstudy and asked to be taken off study before receiving the fourth cycleof HIDAC consolidation; Patient 3001 completed 1 consolidation cycle,withdrew from study and was lost to follow-up; and Patient 3003 received2 cycles of consolidation on study before relapsing. Four patients whocompleted induction received an allogeneic stem cell transplant in CR1(Patients 1002, 1006, 1010, and 3002). Six patients relapsed at a mediantime of 8 months. Among those were Patient 2001 who had not completedinduction and relapsed 7 weeks after diagnosis and Patient 3001 whoreceived only 1 cycle of consolidation therapy, and relapsed 13.5 monthsafter diagnosis.

With a median follow-up of 14.2 months, median event free survival is13.5 months and median OS is 13.6+ months.

6.2. Example 2 CX-01 ODSH Mobilizes Cells of Multiple Lineages from BoneMarrow

Bone marrow biopsies were obtained in the AML, trial described inExample 1.

FIGS. 2A-2C are photomicrographs of biopsies from one of the patients.FIG. 2A is a photomicrograph prior to treatment, and shows the bonemarrow packed with leukemia cells. FIG. 2B is a photograph of bonemarrow at day 14 of the induction cycle, showing elimination of leukemiacells, as expected, and additionally showing an unexpected andsignificant depletion of normal bone marrow cells. FIG. 2C shows thebone marrow at Day 28, showing no evidence of leukemia and restorationof normal bone marrow appearance and function.

The unexpected clearing of the marrow seen in the Day 14 marrow suggeststhat the increased remission rate observed in the current trial can beattributed to ODSH-mediated mobilization of leukemic cells from themarrow into the peripheral circulation, where they became vulnerable tothe infusions of cytarabine and idarubicin. Retention of leukemic cellsin the bone marrow is known to make them more resistant to chemotherapy(Hope et al., Nat. Immunol. 5:738-742 (2004)).

The recovery by Day 28 demonstrates further that the ODSH-mediatedflushing of cells from the marrow does not adversely affect the abilityof the marrow to repopulate and support multi-lineage hematopoiesis.Indeed, the accelerated recovery of platelet and white cell count,consistent with observations from a previous trial in pancreatic cancer,demonstrates that the marrow microenvironments required forthrombopoiesis, erythropoiesis, and granulopoiesis remain healthy.

6.3. Example 4 Phase II Clinical trial for the Treatment of MDS withCX-01 ODSH and Azacitidine

A pilot phase IIa study is conducted to confirm and quantify thetherapeutic effect of adding CX-01 (2-O, 3-O-desulfated heparinderivative) to azacitidine in the treatment of recurrent or refractorymyelodysplastic syndrome.

6.3.1. Primary Objective

The primary objective of the clinical study is to quantify the effect oncomplete response and near complete response rate (CR with incompletecount recovery) after combination therapy with CX-01 and azacitidine inpatients with MDS.

6.3.2. Secondary Objectives

-   -   1. To quantify the partial response rate of combination therapy        with CX-01 and azacitidine in patients with MDS    -   2. To quantify event free, progression free, disease free,        1-year survival, and overall survival of patients treated with        CX-01 and azacitidine    -   3. To quantify hematologic improvement as determined by ANC,        platelet and RBC response    -   4. To characterize and quantify the cytogenetic response as        determined by reversion to normal karyotype

6.3.3. Overall Study Design and Plan Description

A pilot phase IIa, open-label trial is conducted to confirm safety andtherapeutic effect of adding CX-01 to azacitidine in the treatment ofrecurrent or refractory myelodysplastic syndrome. CX-01 is administeredas a 4 mg/kg bolus on Day 1 followed by a continuous intravenousinfusion of 0.25 mg/kg/hr for Days 1 through 5 of each 28-day cycle.Azacitidine is administered at 75 mg/m² as a 15 minute intravenousinfusion daily on Days 1 through 5 of each 28-day cycle.

Patients may continue treatment for up to 6 cycles or until theyexperience unacceptable toxicity that precludes further treatment,disease relapse or progression, and/or at the discretion of theinvestigator. Additional cycles may be administered after consultationwith the Principle Investigator if a clear benefit is demonstrated forthe patient.

A Data and Safety Monitoring Committee meets periodically to review thesafety of the study. Adverse events (AEs) are collected from time ofinformed consent and continue until 30 days after last study treatmentis administered.

6.3.4. Selection of Study Population

Inclusion Criteria: To be eligible to participate in the study, patientsmust meet the following criteria:

-   -   1. Male or female, 18 years of age or older.    -   2. Diagnosis of myelodysplastic syndrome and one of the        following:        -   a. Symptomatic anemia with either hemoglobin<10.0 g/dL or            requiring RBC transfusion        -   b. Thrombocytopenia with a history of two or more platelet            counts<50,000/μL or a significant hemorrhage requiring            platelet transfusions        -   c. Neutropenia with two or more ANC<1,000/μL    -   3. IPSS score of INT-1 or higher at screening    -   4. Patient must have undergone≥4 cycles of prior hypomethylating        agent (decitabine or azacitidine) without response as defined by        IWG criteria or have documented disease progression after prior        response to hypomethylating agent therapy    -   5. ECOG performance status≤2    -   6. >10% disease burden measured by cytomorphology, flow        cytometry, or cytogenetics    -   7. Peripheral white blood cell count<50,000/μL.    -   8. Total bilirubin<1.5×ULN; AST/ALT<2.5×ULN,    -   9. Creatinine<2.0×ULN    -   10. Must be able to understand and willing to sign an        IRB-approved written informed consent document.

Exclusion Criteria: Patients who meet any of the following criteria arenot eligible to participate in the study:

-   -   1. Treatment with any other investigational therapeutic agent        for the treatment of MDS within 7 days prior to study entry    -   2. Presence of significant active infection or congestive heart        failure that is not controlled in the opinion of the        Investigator    -   3. Presence of significant active bleeding    -   4. CNS leukemia    -   5. Positive HIV or hepatitis C serology    -   6. Known allergies, hypersensitivity, or intolerance to any form        of heparin    -   7. Patients receiving any form of anticoagulant therapy (heparin        flushes for IV catheter are permitted)    -   8. Psychiatric or neurologic conditions that could compromise        patient safety or compliance, or interfere with the ability to        give proper informed consent

Withdrawal and Discontinuation of Patients: Patients are free towithdraw consent and/or discontinue participation in the study at anytime, without prejudice to further treatment. A patient's participationin the study may also be discontinued at any time at the discretion ofthe Investigator or Sponsor.

The following may be justifiable reasons for the Investigator or Sponsorto discontinue a patient from treatment:

-   -   The patient was erroneously included in the study (i.e. was        found to be ineligible)    -   The patient experiences an intolerable or unacceptable AE    -   The patient is unable to comply with the requirements of the        protocol    -   The patient participates in another investigational study        without the prior written authorization of the Sponsor or its        designee    -   The patient's participation in the study presents a significant        safety concern.

Patients who experience Grade 4 increases in AST, ALT or bilirubin(e.g., increase in AST or ALT>20×ULN; increase in total bilirubinto>10×ULN) are discontinued from the study, if the Investigator judgesthat the laboratory abnormalities are potentially related to studytreatment. Patients discontinued for this reason are not re-challengedand are followed until resolution of abnormal liver function tests.Patients who are discontinued from study due to an AE are closelymonitored until the resolution or stabilization of the AE. Patients whoreceived at least one dose of study drug and who are discontinued fromtreatment, but not withdrawn from the study, are asked to complete allevaluations for early termination (Early Termination/End of StudyVisit). Patients who discontinue from the study are not replaced.

6.3.5. Treatment Of Patients 6.3.5.1. Treatments Administered

CX-01 is administered as a 4 mg/kg bolus on Day 1 followed by acontinuous intravenous infusion of 0.25 mg/kg/hr for Days 1 through 5 ofeach 28-day cycle. Azacitidine is administered at 75 mg/m² as a 15minute intravenous infusion daily on Days 1 through 5 of each 28-daycycle. Patients may continue treatment for up to 6 cycles or until theyexperience unacceptable toxicity that precludes further treatment,disease relapse or progression, and/or at the discretion of theinvestigator. Additional cycles may be administered after consultationwith the Principle Investigator if a clear benefit is demonstrated forthe patient.

6.3.5.2. Dosing and Method of Administration

Azacitidine and CX-01 doses are calculated based on actual body weightat the beginning of therapy.

Preparation of CX-01 Infusion

The Investigator and pharmacist at the investigational site ensures GoodPharmacy Practices are followed during the preparation of the CX-01 IVsolution. The volumes and CX-01 concentration in the final CX-01 IVsolutions must be verified to be correct based on the patient's actualbody weight measured at the beginning of the cycle.

Preparation of CX-01 Intravenous Bolus Dose

The pharmacist prepares the IV bolus resulting from the 4 mg/kg dosecalculation with the amount of CX-01 from the appropriate number of 2 mLor 10 mL vials. Each 1 mL solution contains 50 mg CX-01 and must befurther diluted in 0.9% sodium chloride. The calculated volume perpatient (based on weight) is added to 30 mL of 0.9% sodium chloridesolution and the total volume administered IV over 5 minutes.

Preparation of CX-01 Continuous Infusion Dose

The pharmacist prepares each study treatment solution, adding thecalculated amounts of CX-01 and 0.9% sodium chloride to an empty,sterile infusion bag. An IV infusion line is then attached to theinfusion bag, and the infusion set purged with the CX-01 solution. ALuer lock (or similar) is then placed at the end of the set. As CX-01doses are weight based, the amount of CX-01 from the vials and salinesolution both vary by patient's weight. Each 1 mL solution contains 50mg CX-01.

For each continuous infusion bag, an appropriate volume andconcentration of CX-01 solution is prepared such that the patientreceives a continuous infusion at the dose of CX-01 of 0.25 mg/kg/hour.The final volume of the CX-01 infusion is 500 to 1000 millimeters/24hours. The infusion bags are prepared at a calculated CX-01concentration based on the patient's actual body weight.

Based upon current stability testing data, CX-01 infusion solutionsexpire at room temperature 72 hours after preparation and should bestored in a refrigerator (2 to 8° C.) until used.

If the IV infusion is interrupted for any reason, the time of infusionstop is recorded, along with the reason. The IV infusion is restarted assoon as possible, and the restart time recorded. The planned cycle daysof treatment administration is not altered, nor is the concentration ofthe CX-01 solution adjusted, to compensate for an interrupted CX-01infusion.

Azacitidine Dose Modifications

For patients with baseline (start of treatment) WBC≥3.0×10⁹/L,ANC≥1.5×10⁹/L, and platelets≥75.0×10⁹/L, adjust the dose as follows,based on nadir counts for any given cycle:

Nadir Counts ANC (×10⁹/L) Platelets (×10⁹/L) % Dose in the Next Course<0.5 <25.0 50% 0.5-1.5 25.0-50.0 67% >1.5 >50.0 100% 

For patients whose baseline counts are WBC<3.0×10⁹/L, ANC<1.5×10⁹/L, orplatelets<75.0×10⁹/L, dose adjustments should be based on nadir countsand bone marrow biopsy cellularity at the time of the nadir as notedbelow, unless there is clear improvement in differentiation (percentageof mature granulocytes is higher and ANC is higher than at onset of thatcourse) at the time of the next cycle, in which case the dose of thecurrent treatment should be continued.

Bone Marrow Biopsy Cellularity at Time of Nadir (%) WBC or PlateletNadir % 30-60 15-30 <15 decrease in counts from baseline % Dose in theNext Course 50-75 > 75 100 50 33 75 50 33

If a nadir as defined in the table above has occurred, the next courseof treatment should be given 28 days after the start of the precedingcourse, provided that both the WBC and the platelet counts are >25%above the nadir and rising. The next course of treatment should be given28 days after the start of the preceding course, provided that both theWBC and the platelet counts are >25% above the nadir and rising. Ifa >25% increase above the nadir is not seen by day 28, counts should bereassessed every 7 days. If a 25% increase is not seen by day 42, thenthe patient should be treated with 50% of the scheduled dose.

CX-01 Dose Modifications

CX-01 is temporarily discontinued in patients who develops aPTT above 45seconds during continuous infusion of CX-01 and at least 8 hours afterthe bolus dose of CX-01, until the aPTT is <35 seconds. CX-01 will thenbe resumed at a 50% dose reduction. If the aPTT rises above 45 secondsat the reduced dose, CX-01 is permanently discontinued. If aPTT at the50% reduced dose is <35 seconds, 4 hours or more after dose reduction,the dose can be escalated by 25%. If the aPTT after dose escalationagain rises above 45 seconds at the reduced dose, the CX-01 istemporarily discontinued until the aPTT is <35 seconds, and then resumedat the previous 50% dose reduction.

Permitted Concomitant Medications

The use of myelopoietic growth factors (G-CSF and GM-CSF) is allowed.

6.3.5.3. Response Criteria

Patients are assessed for response according to the IWG criteria:

Complete Remission (CR)−Defined as <5% myeloblasts with normalmaturation of all cell lines in the bone marrow and peripheral bloodvalues of Hgb>11 g/dL, Platelets>100×10⁹/L, Neutrophils>1.0×10⁹/L, and0% blasts. Persistent dysplasia does not exclude CR but will be noted.

Marrow Complete Response (Marrow CR)—Defined as<5% myeloblasts in thebone marrow and a decrease by>50% from pre-treatment values, but notmeeting the definition of CR above.

Partial Remission (PR)—Defined as meeting the definition of CR abovewith a decrease of myeloblasts in the bone marrow by>50% frompre-treatment values, but absolute myeloblasts still>5%.

Stable Disease (SD)—Defined as not meeting the definitions of CR, MarrowCR, PR, SD, PD, or recurrence/morphologic relapse.

Progressive Disease/Relapse (PD)—Defined as ≥50% increase in blaststo>5% blasts (for patients with less than 5% blasts at baseline only),≥50% increase to >10% blasts (for patients with 5-10% blasts at baselineonly), ≥50% increase to >20% blasts (for patients with 10-20% blasts atbaseline only), ≥50% increase to>30% blasts (for patients with 20-30%blasts at baseline only) or any of the following: At least 50% decrementfrom maximum remission/response in granulocytes or platelets, reductionin Hgb by≥2 g/dL, or New or worsened transfusion dependence not relatedto study drug toxicity. Or for patients with a CR, Marrow CR, or PR asdefined above and subsequently development of one of the following:Return to pre-treatment bone marrow blast percentage, decrement of≥50%from maximum remission/response levels in granulocytes or platelets, orreduction in Hgb concentration by≥1.5 g/dL or transfusion dependence.

Hematologic Improvement

Progressive disease as defined above nullifies hematologic improvement.

Erythroid response requires all of the following (only required ifpre-treatment Hgb<11 g/dL):

-   -   Hgb increase by≥1.5 g/dL    -   Relevant reduction of units of RBC transfusions by an absolute        number of at least 4 RBC transfusions/8 week compared with the        pre-treatment transfusion number in the previous 8 weeks. Only        RBC transfusions given for a Hgb of≤9.0 g/dL pre-treatment will        count in the RBC transfusion response evaluation

Platelet response requires one of the following (only required ifpre-treatment platelets<100×10⁹/L):

-   -   Absolute increase of≥30×10⁹/L (for patients starting        with>20×10⁹/L platelets)    -   Increase from <20×10⁹/L to >20×10⁹/L and absolute increase >100%        (for patients starting with <20×10⁹/L)

Neutrophil response requires the following (only required ifpre-treatment ANC<1.0×10⁹/L):

-   -   At least 100% increase and an absolute increase >0.5×10⁹/L

Cytogenetic Response

Cytogenic Response is defined as reversion to a normal karyotype. Forthis study, reversion of a normal karyotype is defined as no clonalabnormalities detected in a minimum of 20 mitotic cells. Progressivedisease as defined above nullifies cytogenetic response.

6.3.6. Results

Addition of CX-01 ODSH, a CXCL12-interacting heparinoid, improves atleast one of the above-described response criteria (see Section5.4.5.3).

All publications, patents, patent applications and other documents citedin this application are hereby incorporated by reference in theirentireties for all purposes to the same extent as if each individualpublication, patent, patent application or other document wereindividually indicated to be incorporated by reference for all purposes.

While various specific embodiments have been illustrated and described,it will be appreciated that various changes can be made withoutdeparting from the spirit and scope of the invention(s).

1. (canceled)
 2. A method of treating acute myeloid leukemia (AML),comprising: adjunctively administering to a subject with AML who isreceiving a hypomethylating agent a heparinoid that is substantiallydesulfated at the 2-O position and/or 3-O position, in an amount and ata time effective to enhance effectiveness of the hypomethylating agent.3. The method of claim 2, wherein the hypomethylating agent isazacitidine.
 4. The method of claim 2, wherein the heparinoid is atleast 85% desulfated at the 2-O position.
 5. The method of claim 2,wherein the heparinoid is at least 85% desulfated at the 3-O position.6. The method of claim 2, wherein the heparinoid has an averagemolecular weight of about 8 kDa to about 15 kDa.
 7. The method of claim2, wherein the heparinoid is administered subcutaneously.
 8. The methodof claim 2, wherein the heparinoid is administered intravenously.
 9. Themethod of claim 8, wherein the heparinoid is administered as one or morebolus injections.
 10. The method of claim 9, wherein the bolus dose is 2mg/kg-25 mg/kg.
 11. The method of claim 8, wherein the heparinoid isadministered as a continuous infusion.
 12. The method of claim 11,wherein the continuous infusion is administered at a dose of 0.1mg/kg/hr-5 mg/kg/hr.
 13. The method of claim 8, wherein the heparinoidis administered as a bolus injection followed by continuous infusion.14. The method of claim 13, wherein the heparinoid is administered as a4 mg/kg bolus followed by a continuous infusion of 0.25 mg/kg/hr. 15.The method of claim 2, wherein the heparinoid is administered prior tothe hypomethylating agent.
 16. The method of claim 2, wherein theheparinoid is administered in combination with the hypomethylatingagent.
 17. The method of claim 16, wherein the heparinoid isadministered in combination with azacitidine.
 18. The method of claim17, wherein the azacitidine is administered intravenously at a dose of 5mg/m²-500 mg/m².
 19. The method of claim 18, wherein the heparinoid isadministered as a bolus injection at a dose of 4 mg/kg followed by acontinuous intravenous infusion at a dose of 0.25 mg/kg/hr; and whereinazacitidine is administered at a dose of 75 mg/m² as a 15 minuteintravenous infusion.
 20. A method of treating acute myeloid leukemia(AML), comprising: administering azacitidine and a heparinoidsubstantially desulfated at the 2-O position and/or 3-O position to asubject with AML, wherein the azacitidine is administered as anintravenous infusion at a dose of 75 mg/m² as a 15 minute intravenousinfusion daily on Days 1 through 7 of the at least one 28-day cycle; andwherein the heparinoid is administered as a bolus injection at a dose of4 mg/kg on Day 1 followed by a continuous intravenous infusion at a doseof 0.25 mg/kg/hr for Days 1 through 7 of at least one 28-day cycle.